Selective cell targeting using adenovirus and chemical dimers

ABSTRACT

Compositions and methods for retargeting adenovirus to a cell using chemical dimers are described. In particular, a recombinant adenovirus comprising a nucleic acid comprising a capsid-dimerizing agent binder conjugate and a ligand-dimerizing agent binder conjugate is provided.

CROSS REFERENCE TO RELATED APPLICATIONS

This is a continuation of U.S. application Ser. No. 14/485,472, filedSep. 12, 2014, which is a continuation of International Application No.PCT/US2013/031002, filed Mar. 13, 2013, published in English under PCTArticle 21(2), which claims the benefit of U.S. Provisional ApplicationNo. 61/610,416 filed Mar. 13, 2012. The above-listed applications arehereby incorporated by reference in their entirety.

REFERENCE TO SEQUENCE LISTING

The Sequence Listing is submitted as an ASCII text file, created on Jan.22, 2018, 762 KB, which is incorporated by reference herein.

BACKGROUND

Cancer is a debilitating disease that accounts for more than half amillion deaths each year. There is a profound need for more effective,selective and safe treatments for cancer. Existing treatments for thispervasive, life threatening disease, such as chemotherapy and surgery,rarely eliminate all malignant cells, and often exhibit deleteriousside-effects that can outweigh therapeutic benefit.

One approach that has the potential to address many of the shortcomingsof current cancer treatments is oncolytic adenoviral therapy (Pesonen,S. et al., Molecular Pharmaceutics, 8(1): p. 12-28 (2010)). Theseviruses are designed to replicate specifically in cancer cells, butleave normal cells unharmed. One way to engineer tumor selectivity is totarget adenovirus infection to receptors upregulated on tumor cells, forexample EGFR family members (Zhang H, Berezov A, Wang Q, Zhang G, DrebinJ, Murali R, et al. ErbB receptors: from oncogenes to targeted cancertherapies. J Clin Invest. 2007;117(8):2051-8. PMCID: 1934579), CEACAM(Li H J, Everts M, Pereboeva L, Komarova S, Idan A, Curiel D T, et al.Adenovirus tumor targeting and hepatic untargeting by acoxsackie/adenovirus receptor ectodomain anti-carcinoembryonic antigenbispecific adapter. Cancer Res. 2007;67(11):5354-61), EpCAM (Haisma H J,Pinedo H M, Rijswijk A, der Meulen-Muileman I, Sosnowski B A, Ying W, etal. Tumor-specific gene transfer via an adenoviral vector targeted tothe pan-carcinoma antigen EpCAM. Gene Ther. 1999;6(8):1469-7), andHLA-A1/MAGE-A1 (de Vrij J, Uil T G, van den Hengel S K, Cramer S J,Koppers-Lalic D, Verweij M C, et al. Adenovirus targeting toHLA-A1/MAGE-A1-positive tumor cells by fusing a single-chain T-cellreceptor with minor capsid protein IX. Gene Ther. 2008;15(13):978-89).For a review of various strategies of adenovirus targeting, seeNoureddini S C and Curiel D T (Genetic targeting strategies foradenovirus. Mol Pharm. 2005;2(5):341-7; Nicklin S A, Wu E, Nemerow G R,Baker A H. The influence of adenovirus fiber structure and function onvector development for gene therapy. Mol Ther. 2005;12(3):384-93).

Adenovirus (Ad) is a self-replicating biological machine. It consists ofa linear double-stranded 36 kb DNA genome sheathed in a protein coat. Adrequires a human host cell to replicate. It invades and hijacks thecellular replicative machinery to reproduce and upon assembly induceslytic cell death to escape the cell and spread and invade surroundingcells (FIG. 1). No ab initio system has come close to mimicking theautonomy and efficiency of Ad, however, Applicants have developed newstrategies to systematically manipulate the Ad genome to create noveladenoviruses. Henceforth, with the ability to manipulate the Ad genome,Applicants can take the virus by the horns and redesign it to performthe functions of tumor-specific infection, replication, and cellkilling.

Currently, adenoviral vectors rely on a single cellular receptor fortheir uptake, which significantly limits their therapeutic potential.Ad5 infection is mediated primarily through interactions between thefiber protein on the outer viral capsid and the coxsackie and adenovirusreceptor (CAR) on human epithelial cells. Unfortunately, many cancercells do not express CAR, such as mesenchymal and deadly metastatictumor cells. Since viral replication/killing is limited by the abilityto infect cells, there is a need for viruses that infect tumor cells viareceptors other than CAR, ideally those specifically upregulated ontumor cells. The present invention addresses these and other needs inthe art by providing viral compositions and methods that chemically linkviral capsids via chemical adapters to a broad variety of cellularreceptors. Provided herein is a novel, inducible, genetically encodedchemical adapter system that retargets infection to multiple cell types,and is not lost upon viral replication. The compositions provided hereincan be used to customize an oncolytic virus to target different cellularreceptors over the course of infection.

SUMMARY

In one aspect, a recombinant nucleic acid encoding a capsid-dimerizingagent binder conjugate and a ligand-dimerizing agent binder conjugateare provided.

In another aspect, a recombinant adenovirus including a recombinantnucleic acid provided herein including embodiments thereof is provided.

In another aspect, a recombinant adenovirus including acapsid-dimerizing agent binder conjugate is provided.

In another aspect, a cell including a recombinant adenovirus providedherein including embodiments thereof is provided.

In another aspect, a method of forming an adenoviral cancer celltargeting construct is provided. The method includes infecting a cellwith a recombinant adenovirus provided herein, thereby forming anadenoviral infected cell. The adenoviral infected cell is allowed toexpress the recombinant nucleic acid, thereby forming aligand-dimerizing agent binder conjugate and a recombinant adenovirusincluding a capsid-dimerizing agent binder conjugate. The recombinantadenovirus and the ligand-dimerizing agent binder conjugate arecontacted with a dimerizing agent. The recombinant adenovirus and theligand-dimerizing agent binder conjugate are allowed to bind to thedimerizing agent, thereby forming the adenoviral cancer cell targetingconstruct.

In another aspect, a method of targeting a cell is provided. The methodincludes contacting a cell with a recombinant adenovirus provided hereinincluding embodiments thereof.

In another aspect, a method of targeting a cancer cell in a cancerpatient is provided. The method includes administering to a cancerpatient a recombinant adenovirus provided herein. The recombinantadenovirus is allowed to infect a cell in the cancer patient, therebyforming an adenoviral infected cell. The adenoviral infected cell isallowed to express the recombinant nucleic acid, thereby forming aligand-dimerizing agent binder conjugate and a recombinant adenovirusincluding a capsid-dimerizing agent binder conjugate. The cancer patientis administered with a dimerizing agent. The recombinant adenovirus andthe ligand-dimerizing agent binder conjugate are allowed to bind to thedimerizing agent, thereby forming an adenoviral cancer cell targetingconstruct. The adenoviral cancer cell targeting construct is allowed tobind to a cancer cell, thereby targeting the cancer cell in the cancerpatient.

In another aspect, a method of targeting a cell is provided. The methodincludes contacting a first cell with a recombinant adenovirus providedherein. The recombinant adenovirus is allowed to infect the first cell,thereby forming an adenoviral infected cell. The adenoviral infectedcell is allowed to express the recombinant nucleic acid, thereby forminga ligand-dimerizing agent binder conjugate and a recombinant adenoviruscomprising a capsid-dimerizing agent binder conjugate. Theligand-dimerizing agent binder conjugate and the recombinant adenovirusare contacted with a dimerizing agent. The recombinant adenovirus andthe ligand-dimerizing agent binder conjugate are allowed to bind to thedimerizing agent, thereby forming an adenoviral cell targetingconstruct. The adenoviral cell targeting construct is allowed to bind toa second cell, thereby targeting the cell.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. General rationale of oncolytic viral cancer therapy.

FIG. 2. Structural features of adenovirus and a map of the adenovirusgenome with transcriptional units in boxes and labeled genes.

FIGS. 3A-3B. Outline of the Adsembly and Ad-SlicR adenovirus genomemanipulation strategies developed by Applicants. FIG. 3A upper panel:The Ad genome is organized into early (E1-4) and late (L1-5)transcription units that express multiple genes via alternativesplicing. Arrows represent multi-gene transcriptional units used by theadenovirus with functional organization reminiscent of operons. Thegenome is split into transcriptional and functional units (‘parts’) andcloned into plasmids (FIG. 3A lower panel). The Library of partsincludes mutants, alternate serotypes and transgenes. Systematicmulti-site specific in vitro re-assembly (Adsembly or Ad-SLIC) andreconstitution of virus is performed. FIG. 3B: the Adenovirus genome(FIG. 3B top panel) is separated into components (FIG. 3B second panelfrom top). Mutagenesis is performed on individual vectors to buildlibrary parts (FIG. 3B third panel form the top) and the virus isassembled in vitro (FIG. 3B bottom panel) to generate noveladenoviruses.

FIG. 4. Ribbon representation of the adenovirus fiber protein trimer.The N terminus (left) is bound to the surface of the capsid, with theC-terminal knob domain farthest away from the virus core. The flexibleH1 loop the knob domain has been used for peptides insertions to impartnew properties to fiber.

FIG. 5. Structure of immunosuppressive anti-tumor drug and antibioticrapamycin and rapalog AP21967.

FIG. 6. Genome assembly strategy utilizing the building and combinationof components to systematically create combination mutations in noveladenoviruses.

FIGS. 7A-7B. Ad 122 is a viable adenovirus expressing fiber with the FRBinsertion. Ad-122 is a viable adenovirus expressing fiber with the FRBinsertion. FIG. 7A: Western blot using anti-fiber antibody 4D2 (Abcam)on lysates from Ad-122 and Wt Ad5 infected 293 E4 cells 48 h p.i. FIG.7B: Bright field and GFP fluorescence images of 293 E4 cells infectedwith Ad-122 48 h p.i. showing significant CPE.

FIGS. 8A-8E. Genetic configurations to express FRB-Fiber and FKBP fromAd5 E3 region. FIG. 8A: Wild-type Ad5 E3 region. FIG. 8B: FRB insertioninto fiber gene. FIG. 8C: Co-translational expression of FKBP usingFurin-2A auto-cleavage sequence. FIG. 8D: Co-transcriptional expressionof FKBP using IRES element on fiber transcript. FIG. 8E: Replacement ofE3B encoded proteins (RIDα, RIDβ, 14.7 k) with FKBP.

FIG. 9. AD-178 expresses FKBP during infection. Lysates collected frominfected 293 E4 cells 24 and 60 h p.i. and probed with anti-fiber (toppanel of FIG. 9) and anti-FKBP antibody ab2918 (Abcam; bottom panel ofFIG. 9).

FIGS. 10A-10B. Ribbon model of FRB-fiber knob-domain in complex withrapamycin/VHH-FKBP and VHH target. Ad5 knob trimer (PDB ID 1KNB) withFRB domain in complex with FKBP (PDB ID 1NSG) as a C-terminal fusion ofVHH, binding its target (PDB ID 3EBA). FIG. 10A: Model from ‘top down’view. FIG. 10B: Model from ‘side’ view, showing that the bindinginterface of the VHH is facing away from the virus particle if it isfused to the N-terminus of FKBP.

FIG. 11. Immunofluorescence to detect fiber and CEAVHH-FKBP localizationin infected 293 E4 cells. 293 E4 cells infected with either Ad-177(CEAVHH-FKBP, FRB-fiber) or Ad-199 (CEAVHH-FKBP, wt fiber) and 500 nMrap or solvent only (EtOH) added 30 h p.i. Cells fixed at 36 h andstained with anti-fiber antibody 4D2 or anti-FBKP antibody ab2918(Abcam).

FIG. 12. FKBP fusion protein does not detectibly accumulate whencontrolled by 5′ IRES on fiber gene. 293 E4 cells infected withrecombinant adenoviruses. Cells harvested, and soluble proteins probedfor fiber and FKBP expression by immunoblot. Top panel: FRB-fiberaccumulates during infection. Bottom panel: VHH-FKBP (˜32 kDa) is notdetectible.

FIG. 13. Representative IMAGEXPRESS™ images of rapamycin-inducedEGFR-retargeted Ad5 infection of MDA MB 468. Ad-178 expressing aGFP-reporter was prepared in the presence or absence of 500 nM rapamycinby infection of 293 E4 cells, and supernatant was used to infect MDA MB468 in culture. FIG. 13 left panel represents infections with undilutedviral supernatant; FIG. 13 right panel represents infections with 1/16dilution of viral supernatant.

FIG. 14. Representative IMAGEXPRESS™ images of rapamycin-inducedEGFR-retargeted Ad5 infection of MDA MB 453. Ad-178 expressing aGFP-reporter was prepared in the presence or absence of 500 nM rapamycinby infection of 293 E4 cells, and supernatant was used to infect MDA MB453 in culture. FIG. 14 left panel represents infections with undilutedviral supernatant; FIG. 14 right panel represents infections with ⅛dilution of viral supernatant.

FIG. 15. Representative IMAGEXPRESS™ images of rapamycin-inducedEGFR-retargeted Ad5 infection of MDA MB 231. Ad-178 expressing aGFP-reporter was prepared in the presence or absence of 500 nM rapamycinby infection of 293 E4 cells, and supernatant was used to infect MDA MB231 in culture. FIG. 15 left panel represents infections with undilutedviral supernatant; FIG. 15 right panel represents infections with ⅛dilution of viral supernatant.

FIG. 16. Representative IMAGEXPRESS™ images of rapamycin-inducedEGFR-retargeted Ad5 infection of HS578T. Ad-178 expressing aGFP-reporter was prepared in the presence or absence of 500 nM rapamycinby infection of 293 E4 cells, and supernatant was used to infect HS578Tin culture. FIG. 16 left panel represents infections with undilutedviral supernatant; FIG. 16 right panel represents infections with ¼dilution of viral supernatant.

FIG. 17. Representative IMAGEXPRESS™ images of rapamycin-inducedEGFR-retargeted Ad5 infection of U87. Ad-178 expressing a GFP-reporterwas prepared in the presence or absence of 500 nM rapamycin by infectionof 293 E4 cells, and supernatant was used to infect U87 in culture. FIG.17 left panel represents infections with undiluted viral supernatant;FIG. 17 right panel represents infections with ⅛ dilution of viralsupernatant.

FIG. 18. Infection of a panel of breast cancer cell lines byrapamycin-induced EGFR-retargeted adenovirus. Ad-178 expressing aGFP-reporter was prepared in the presence or absence of 500 nM rapamycinby infection of 293 E4 cells, and supernatant was diluted 50-fold usedto infect cells in culture. % infected cells determined 24 h p.i. byIMAGEXPRESS™ analysis of GFP positive nuclei. Each pair of columns inthe histogram shows infection of a breast cancer cell line with Ad-178expressing a GFP-reporter prepared in the absence (left column) or inthe presence (right column) of rapamycin. The histogram shows from leftto right infection of MDA MB468 cells (90% without rapamycin; 96% plusrapamycin), MDA MB415 cells (69% without rapamycin; 55% plus rapamycin),MDA MB453 (16% without rapamycin; 73% plus rapamycin), MDA MB231 (16%without rapamycin; 78% plus rapamycin), BTS49 (37% without rapamycin;74% plus rapamycin), and HS578 (0% without rapamycin; 28% plusrapamycin), respectively.

FIG. 19. Infection of a panel of cancer cell lines by rapamycin-inducedEGFR-retargeted adenovirus. An Ad-178 expressing a GFP-reporter wasprepared in the presence or absence of 500 nM rapamycin by infection of293 E4 cells, and supernatant was diluted 50-fold used to infectdifferent cancer cells in culture. % infected cells determined 24 h p.i.by IMAGEXPRESS™ analysis of GFP positive nuclei. Each pair of columns inthe histogram shows infection of a cancer cell line with Ad-178expressing a GFP-reporter prepared in the absence (left column) or inthe presence (right column) of rapamycin. The histogram shows from leftto right infection of U2OS osteosarcoma cell line (52% withoutrapamycin; 24% plus rapamycin), H1299 lung carcinoma cell line (78%without rapamycin; 78% plus rapamycin), A549 lung carcinoma cell line(37% without rapamycin; 66% plus rapamycin), and U87 glioblastoma cellline (11% without rapamycin; 50% plus rapamycin), respectively.

FIG. 20. Rapamycin concentration optimization for EGFR-retargeting withAd-178 to infect MDA MB 453. Ad-178 expressing a GFP-reporter wasprepared in the presence or absence of various rapamycin concentrationduring infection of 293 E4 cells, and supernatant was used to infect MDAMB 453 cells in culture. % infected cells determined 24 h p.i. by FACSanalysis of GFP positive cells. Percent GFP positive cells were 54.13%at 0 nM rap, 58.96% at 10 nM rap, 68.23% at 25 nM rap, 76.75% at 50 nMrap, 70.73% at 100 nM rap, and 71.76% at 500 nM rap, respectively.

FIGS. 21A-21C. EGFR-dependent infection of Ad-178. Infection quantifiedby FACS, counting cells expressing adenovirus-delivered GFP gene, >30 kevents each. FIG. 21A: Adenovirus with genetically encoded FRB domaininsertion in fiber, and EGFRVHH-FKBP fusion protein prepared in thepresence or absence of 50 nM rapamycin and used to infect MDA MB 453cells with or without shRNA-mediated EGFR knockdown. FIG. 21B:Adenovirus with only genetically encoded FRB domain insertion in fiber,prepared in the presence or absence of 50 nM rapamycin and used toinfect MDA MB 453 cells with or without shRNA-mediated EGFR knockdown.FIG. 21C: Verification of stable, shRNA-mediated EGFR knockdown in MDAMB 453 cells by protein immunoblot.

FIG. 22. Rapamycin induced EGFR-retargeting of Ad-178 enhances cellkilling of HS578T. CPE assay using WST-1 reagent for % metabolicactivity vs. uninfected cells 9 days post infection. 50 nM rapamycinadded to cells at time points indicated in figure legend. Data pointsshown are averages of samples in triplicate.

FIGS. 23A-23H. Targeted infection of cell lines by control Ad, or by Adencoding ligands fused to FKBP. The viruses encoded either the CEACAMsingle domain antibody fragment fused to FKBP (CEAVHH-FKBP), the EGFRsingle domain antibody fragment fused to FKBP (EGFRVHH-FKBP), or domain4 of protective antigen fused to FKBP (D4-FKBP). The adenoviruses wereprepared in the presence or absence of 100 nM rapamycin by infection of293 E4 cells, and supernatant was used to infect the targeted celllines: FIG. 23A shows infection of MDA MB231. FIG. 23B shows infectionof MDA MB453. FIG. 23C shows infection of MDA MB468. FIG. 23D showsinfection of HS578T. FIG. 23E shows infection of BT474. FIG. 23F showsinfection of MCF7. FIG. 23G shows infection of CHO K1. FIG. 23H showsinfection of CHO R1.1. Numbers on top of the columns represent % of GFP(i.e. infected) cells.

FIGS. 24A-24D. Targeted infection of cell lines using AP21967 and mutantFRB domain-containing Ad. The adenoviruses were prepared in the presenceor absence of 100 nM rapamycin or 100 nM AP21967 by infection of 293 E4cells, and supernatant was used to infect the targeted cell lines. FIG.24A shows infection of MDA MB453. FIG. 24B shows infection of MDA MB468.FIG. 24C shows infection of MDA HS578T. FIG. 24D shows infection of MDAMCF7. Numbers on top of the columns represent % of GFP (i.e. infected)cells.

FIG. 25. Targeted infection of cell lines using AP21967 and mutant FRBdomain-containing Ad. The EGFR-targeted adenovirus containing theFRB-mutant in the capsid was prepared with a range of concentration ofAP21967 or 100 nM rapamycin were prepared by infection of 293 E4 cells,and supernatant was used to infect the MDA MB 453. Numbers on top of thecolumns represent % of GFP (i.e. infected) cells.

FIG. 26. Targeted infection of cell lines ectopically expressedligand-FKBP fusion, EGFRVHH-FKBP. The ligand-FKBP fusion (or GFP as acontrol) was transiently expressed in 293 E4 cells, and infected withAd-122. The virus was prepared in the presence of absence of 100 nMrapamycin, and the supernatant was used to infect the MDA MB 231.Numbers on top of the columns represent % of GFP (i.e. infected) cells.

FIGS. 27A-27C. Targeted infection of cell lines by control Ad, or by Adencoding ligands fused to FKBP. The adenoviruses were prepared in thepresence or absence of 100 nM rapamycin by infection of 293 E4 cells,and supernatant was used to infect the targeted cell lines. FIG. 27Aupper panel shows infection of MDA MB231. FIG. 27A middle panel showsinfection of MDA MB453. FIG. 27A lower panel shows infection of MDAMB468. FIG. 27B upper panel shows infection of HS578T. FIG. 27B middlepanel shows infection of BT474. FIG. 27B lower panel shows infection ofMCF7. FIG. 27C upper panel shows infection of CHO K1. FIG. 27C lowerpanel shows infection of CHO R1.1. Numbers on top of the columnsrepresent % of GFP (i.e. infected) cells.

DETAILED DESCRIPTION I. Definitions

“Nucleic acid” refers to deoxyribonucleotides or ribonucleotides andpolymers thereof in either single- or double-stranded form, andcomplements thereof. The term encompasses nucleic acids containing knownnucleotide analogs or modified backbone residues or linkages, which aresynthetic, naturally occurring, and non-naturally occurring, which havesimilar binding properties as the reference nucleic acid, and which aremetabolized in a manner similar to the reference nucleotides. Examplesof such analogs include, without limitation, phosphorothioates,phosphoramidates, methyl phosphonates, chiral-methyl phosphonates,2-O-methyl ribonucleotides, peptide-nucleic acids (PNAs).

The terms “Ad5” and “Adenoviral genome” as used herein refer to thenucleic sequence as set forth in SEQ ID NO: 108.

Unless otherwise indicated, a particular nucleic acid sequence alsoimplicitly encompasses conservatively modified variants thereof (e.g.,degenerate codon substitutions) and complementary sequences, as well asthe sequence explicitly indicated. Specifically, degenerate codonsubstitutions may be achieved by generating sequences in which the thirdposition of one or more selected (or all) codons is substituted withmixed-base and/or deoxyinosine residues (Batzer et al., Nucleic AcidRes. 19:5081 (1991); Ohtsuka et al., J. Biol. Chem. 260:2605-2608(1985); Rossolini et al., Mol. Cell. Probes 8:91-98 (1994)). The termnucleic acid is used interchangeably with gene, cDNA, mRNA,oligonucleotide, and polynucleotide.

A particular nucleic acid sequence also implicitly encompasses “splicevariants.” Similarly, a particular protein encoded by a nucleic acidimplicitly encompasses any protein encoded by a splice variant of thatnucleic acid. “Splice variants,” as the name suggests, are products ofalternative splicing of a gene. After transcription, an initial nucleicacid transcript may be spliced such that different (alternate) nucleicacid splice products encode different polypeptides. Mechanisms for theproduction of splice variants vary, but include alternate splicing ofexons. Alternate polypeptides derived from the same nucleic acid byread-through transcription are also encompassed by this definition. Anyproducts of a splicing reaction, including recombinant forms of thesplice products, are included in this definition. An example ofpotassium channel splice variants is discussed in Leicher, et al., J.Biol. Chem. 273(52):35095-35101 (1998).

Construction of suitable vectors containing the desired therapeutic genecoding and control sequences may employ standard ligation andrestriction techniques, which are well understood in the art (seeManiatis et al., in Molecular Cloning: A Laboratory Manual, Cold SpringHarbor Laboratory, New York (1982)). Isolated plasmids, DNA sequences,or synthesized oligonucleotides may be cleaved, tailored, and re-ligatedin the form desired.

Nucleic acid is “operably linked” when it is placed into a functionalrelationship with another nucleic acid sequence. For example, DNA for apresequence or secretory leader is operably linked to DNA for apolypeptide if it is expressed as a preprotein that participates in thesecretion of the polypeptide; a promoter or enhancer is operably linkedto a coding sequence if it affects the transcription of the sequence; ora ribosome binding site is operably linked to a coding sequence if it ispositioned so as to facilitate translation. Generally, “operably linked”means that the DNA sequences being linked are near each other, and, inthe case of a secretory leader, contiguous and in reading phase.However, enhancers do not have to be contiguous. Linking is accomplishedby ligation at convenient restriction sites. If such sites do not exist,the synthetic oligonucleotide adaptors or linkers are used in accordancewith conventional practice.

The terms “identical” or percent “identity,” in the context of two ormore nucleic acids or polypeptide sequences, refer to two or moresequences or subsequences that are the same or have a specifiedpercentage of amino acid residues or nucleotides that are the same(i.e., about 60% identity, preferably 65%, 70%, 75%, 80%, 85%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher identity over aspecified region, when compared and aligned for maximum correspondenceover a comparison window or designated region) as measured using a BLASTor BLAST 2.0 sequence comparison algorithms with default parametersdescribed below, or by manual alignment and visual inspection (see,e.g., NCBI web site or the like). Such sequences are then said to be“substantially identical.” This definition also refers to, or may beapplied to, the compliment of a test sequence. The definition alsoincludes sequences that have deletions and/or additions, as well asthose that have substitutions. As described below, the preferredalgorithms can account for gaps and the like. Preferably, identityexists over a region that is at least about 25 amino acids ornucleotides in length, or more preferably over a region that is 50-100amino acids or nucleotides in length.

For sequence comparison, typically one sequence acts as a referencesequence, to which test sequences are compared. When using a sequencecomparison algorithm, test and reference sequences are entered into acomputer, subsequence coordinates are designated, if necessary, andsequence algorithm program parameters are designated. Preferably,default program parameters can be used, or alternative parameters can bedesignated. The sequence comparison algorithm then calculates thepercent sequence identities for the test sequences relative to thereference sequence, based on the program parameters.

A “comparison window”, as used herein, includes reference to a segmentof any one of the number of contiguous positions selected from the groupconsisting of from 20 to 600, usually about 50 to about 200, moreusually about 100 to about 150 in which a sequence may be compared to areference sequence of the same number of contiguous positions after thetwo sequences are optimally aligned. Methods of alignment of sequencesfor comparison are well-known in the art. Optimal alignment of sequencesfor comparison can be conducted, e.g., by the local homology algorithmof Smith & Waterman, Adv. Appl. Math. 2:482 (1981), by the homologyalignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443 (1970),by the search for similarity method of Pearson & Lipman, Proc. Nat'l.Acad. Sci. USA 85:2444 (1988), by computerized implementations of thesealgorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin GeneticsSoftware Package, Genetics Computer Group, 575 Science Dr., Madison,Wis.), or by manual alignment and visual inspection (see, e.g., CurrentProtocols in Molecular Biology (Ausubel et al., eds. 1995 supplement)).

A preferred example of algorithm that is suitable for determiningpercent sequence identity and sequence similarity are the BLAST andBLAST 2.0 algorithms, which are described in Altschul et al., Nuc. AcidsRes. 25:3389-3402 (1977) and Altschul et al., J. Mol. Biol. 215:403-410(1990), respectively. BLAST and BLAST 2.0 are used, with the parametersdescribed herein, to determine percent sequence identity for the nucleicacids and proteins of the invention. Software for performing BLASTanalyses is publicly available through the National Center forBiotechnology Information, as known in the art. This algorithm involvesfirst identifying high scoring sequence pairs (HSPs) by identifyingshort words of length W in the query sequence, which either match orsatisfy some positive-valued threshold score T when aligned with a wordof the same length in a database sequence. T is referred to as theneighborhood word score threshold (Altschul et al., supra). Theseinitial neighborhood word hits act as seeds for initiating searches tofind longer HSPs containing them. The word hits are extended in bothdirections along each sequence for as far as the cumulative alignmentscore can be increased. Cumulative scores are calculated using, fornucleotide sequences, the parameters M (reward score for a pair ofmatching residues; always >0) and N (penalty score for mismatchingresidues; always <0). For amino acid sequences, a scoring matrix is usedto calculate the cumulative score. Extension of the word hits in eachdirection are halted when: the cumulative alignment score falls off bythe quantity X from its maximum achieved value; the cumulative scoregoes to zero or below, due to the accumulation of one or morenegative-scoring residue alignments; or the end of either sequence isreached. The BLAST algorithm parameters W, T, and X determine thesensitivity and speed of the alignment. The BLASTN program (fornucleotide sequences) uses as defaults a wordlength (W) of 11, anexpectation (E) of 10, M=5, N=−4 and a comparison of both strands. Foramino acid sequences, the BLASTP program uses as defaults a wordlengthof 3, and expectation (E) of 10, and the BLOSUM62 scoring matrix (seeHenikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89:10915 (1989))alignments (B) of 50, expectation (E) of 10, M=5, N=−4, and a comparisonof both strands.

The terms “polypeptide,” “peptide” and “protein” are usedinterchangeably herein to refer to a polymer of amino acid residues. Theterms apply to amino acid polymers in which one or more amino acidresidue is an artificial chemical mimetic of a corresponding naturallyoccurring amino acid, as well as to naturally occurring amino acidpolymers and non-naturally occurring amino acid polymer.

The term “amino acid” refers to naturally occurring and synthetic aminoacids, as well as amino acid analogs and amino acid mimetics thatfunction in a manner similar to the naturally occurring amino acids.Naturally occurring amino acids are those encoded by the genetic code,as well as those amino acids that are later modified, e.g.,hydroxyproline, γ-carboxyglutamate, and O-phosphoserine. Amino acidanalogs refers to compounds that have the same basic chemical structureas a naturally occurring amino acid, i.e., an a carbon that is bound toa hydrogen, a carboxyl group, an amino group, and an R group, e.g.,homoserine, norleucine, methionine sulfoxide, methionine methylsulfonium. Such analogs have modified R groups (e.g., norleucine) ormodified peptide backbones, but retain the same basic chemical structureas a naturally occurring amino acid. Amino acid mimetics refers tochemical compounds that have a structure that is different from thegeneral chemical structure of an amino acid, but that functions in amanner similar to a naturally occurring amino acid.

Amino acids may be referred to herein by either their commonly knownthree letter symbols or by the one-letter symbols recommended by theIUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise,may be referred to by their commonly accepted single-letter codes.

“Conservatively modified variants” applies to both amino acid andnucleic acid sequences. With respect to particular nucleic acidsequences, conservatively modified variants refers to those nucleicacids which encode identical or essentially identical amino acidsequences, or where the nucleic acid does not encode an amino acidsequence, to essentially identical sequences. Because of the degeneracyof the genetic code, a large number of functionally identical nucleicacids encode any given protein. For instance, the codons GCA, GCC, GCGand GCU all encode the amino acid alanine. Thus, at every position wherean alanine is specified by a codon, the codon can be altered to any ofthe corresponding codons described without altering the encodedpolypeptide. Such nucleic acid variations are “silent variations,” whichare one species of conservatively modified variations. Every nucleicacid sequence herein which encodes a polypeptide also describes everypossible silent variation of the nucleic acid. One of skill willrecognize that each codon in a nucleic acid (except AUG, which isordinarily the only codon for methionine, and TGG, which is ordinarilythe only codon for tryptophan) can be modified to yield a functionallyidentical molecule. Accordingly, each silent variation of a nucleic acidwhich encodes a polypeptide is implicit in each described sequence withrespect to the expression product, but not with respect to actual probesequences.

As to amino acid sequences, one of skill will recognize that individualsubstitutions, deletions or additions to a nucleic acid, peptide,polypeptide, or protein sequence which alters, adds or deletes a singleamino acid or a small percentage of amino acids in the encoded sequenceis a “conservatively modified variant” where the alteration results inthe substitution of an amino acid with a chemically similar amino acid.Conservative substitution tables providing functionally similar aminoacids are well known in the art. Such conservatively modified variantsare in addition to and do not exclude polymorphic variants, interspecieshomologs, and alleles of the invention.

The following eight groups each contain amino acids that areconservative substitutions for one another: 1) Alanine (A), Glycine (G);2) Aspartic acid (D), Glutamic acid (E); 3) Asparagine (N), Glutamine(Q); 4) Arginine (R), Lysine (K); 5) Isoleucine (I), Leucine (L),Methionine (M), Valine (V); 6) Phenylalanine (F), Tyrosine (Y),Tryptophan (W); 7) Serine (S), Threonine (T); and 8) Cysteine (C),Methionine (M) (see, e.g., Creighton, Proteins (1984)).

The term “recombinant” when used with reference, e.g., to a cell, virus,nucleic acid, protein, or vector, indicates that the cell, virus,nucleic acid, protein or vector, has been modified by the introductionof a heterologous nucleic acid or protein or the alteration of a nativenucleic acid or protein, or that the cell is derived from a cell somodified. Thus, for example, recombinant cells express genes that arenot found within the native (non-recombinant) form of the cell orexpress native genes that are otherwise abnormally expressed, underexpressed or not expressed at all.

The phrase “stringent hybridization conditions” refers to conditionsunder which a probe will hybridize to its target subsequence, typicallyin a complex mixture of nucleic acids, but to no other sequences.Stringent conditions are sequence-dependent and will be different indifferent circumstances. Longer sequences hybridize specifically athigher temperatures. An extensive guide to the hybridization of nucleicacids is found in Tijssen, Techniques in Biochemistry and MolecularBiology—Hybridization with Nucleic Probes, “Overview of principles ofhybridization and the strategy of nucleic acid assays” (1993).Generally, stringent conditions are selected to be about 5-10° C. lowerthan the thermal melting point (T_(m)) for the specific sequence at adefined ionic strength pH. The T_(m) is the temperature (under definedionic strength, pH, and nucleic concentration) at which 50% of theprobes complementary to the target hybridize to the target sequence atequilibrium (as the target sequences are present in excess, at T_(m),50% of the probes are occupied at equilibrium). Stringent conditions mayalso be achieved with the addition of destabilizing agents such asformamide. For selective or specific hybridization, a positive signal isat least two times background, preferably 10 times backgroundhybridization. Exemplary stringent hybridization conditions can be asfollowing: 50% formamide, 5×SSC, and 1% SDS, incubating at 42° C., or,5×SSC, 1% SDS, incubating at 65° C., with wash in 0.2×SSC, and 0.1% SDSat 65° C.

Nucleic acids that do not hybridize to each other under stringentconditions are still substantially identical if the polypeptides whichthey encode are substantially identical. This occurs, for example, whena copy of a nucleic acid is created using the maximum codon degeneracypermitted by the genetic code. In such cases, the nucleic acidstypically hybridize under moderately stringent hybridization conditions.Exemplary “moderately stringent hybridization conditions” include ahybridization in a buffer of 40% formamide, 1 M NaCl, 1% SDS at 37° C.,and a wash in 1×SSC at 45° C. A positive hybridization is at least twicebackground. Those of ordinary skill will readily recognize thatalternative hybridization and wash conditions can be utilized to provideconditions of similar stringency. Additional guidelines for determininghybridization parameters are provided in numerous reference, e.g., andCurrent Protocols in Molecular Biology, ed. Ausubel, et al., John Wiley& Sons.

For PCR, a temperature of about 36° C. is typical for low stringencyamplification, although annealing temperatures may vary between about32° C. and 48° C. depending on primer length. For high stringency PCRamplification, a temperature of about 62° C. is typical, although highstringency annealing temperatures can range from about 50° C. to about65° C., depending on the primer length and specificity. Typical cycleconditions for both high and low stringency amplifications include adenaturation phase of 90° C.-95° C. for 30 sec −2 min., an annealingphase lasting 30 sec. −2 min., and an extension phase of about 72° C.for 1-2 min. Protocols and guidelines for low and high stringencyamplification reactions are provided, e.g., in Innis et al. (1990) PCRProtocols, A Guide to Methods and Applications, Academic Press, Inc.N.Y.).

The terms “transfection”, “transduction”, “transfecting” or“transducing” can be used interchangeably and are defined as a processof introducing a nucleic acid molecule or a protein to a cell. Nucleicacids are introduced to a cell using non-viral or viral-based methods.The nucleic acid molecule can be a sequence encoding complete proteinsor functional portions thereof. Typically, a nucleic acid vector,comprising the elements necessary for protein expression (e.g., apromoter, transcription start site, etc.). Non-viral methods oftransfection include any appropriate method that does not use viral DNAor viral particles as a delivery system to introduce the nucleic acidmolecule into the cell. Exemplary non-viral transfection methods includecalcium phosphate transfection, liposomal transfection, nucleofection,sonoporation, transfection through heat shock, magnetifection andelectroporation. For viral-based methods, any useful viral vector can beused in the methods described herein. Examples of viral vectors include,but are not limited to retroviral, adenoviral, lentiviral andadeno-associated viral vectors. In some aspects, the nucleic acidmolecules are introduced into a cell using a adenoviral vector followingstandard procedures well known in the art. The terms “transfection” or“transduction” also refer to introducing proteins into a cell from theexternal environment. Typically, transduction or transfection of aprotein relies on attachment of a peptide or protein capable of crossingthe cell membrane to the protein of interest. See, e.g., Ford et al.(2001) Gene Therapy 8:1-4 and Prochiantz (2007) Nat. Methods 4:119-20.

Expression of a transfected gene can occur transiently or stably in ahost cell. During “transient expression” the transfected nucleic acid isnot integrated into the host cell genome, and is not transferred to thedaughter cell during cell division. Since its expression is restrictedto the transfected cell, expression of the gene is lost over time. Incontrast, stable expression of a transfected gene can occur when thegene is co-transfected with another gene that confers a selectionadvantage to the transfected cell. Such a selection advantage may be aresistance towards a certain toxin that is presented to the cell.Expression of a transfected gene can further be accomplished bytransposon-mediated insertion into to the host genome. Duringtransposon-mediated insertion, the gene is positioned in a predictablemanner between two transposon linker sequences that allow insertion intothe host genome as well as subsequent excision.

“FKBP” or an “FKBP protein or polypeptide” as referred to hereinincludes any of the naturally-occurring forms of the FKBP protein, orvariants thereof that maintain FKBP protein activity (e.g. within atleast 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity comparedto FKBP). In some embodiments, variants have at least 90%, 95%, 96%,97%, 98%, 99% or 100% amino acid sequence identity across the wholesequence or a portion of the sequence (e.g. a 50, 100, 150 or 200continuous amino acid portion) compared to a naturally occurring FKBPprotein as set forth in SEQ ID NO:66.

“FRB” or an “FRB protein or polypeptide” as referred to herein includesany of the naturally-occurring forms of the FRB protein, or variantsthereof that maintain FRB protein activity (e.g. within at least 50%,80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity compared to FRB). Insome embodiments, variants have at least 90%, 95%, 96%, 97%, 98%, 99% or100% amino acid sequence identity across the whole sequence or a portionof the sequence (e.g. a 50, 100, 150 or 200 continuous amino acidportion) compared to a naturally occurring FRB protein as set forth inSEQ ID NO:69.

“EGFR” refers to the epidermal growth factor receptor corresponding tothe amino acid sequence as set forth in SEQ ID NO:21.

“VHH” refers to a single domain antibody consisting of a singlemonomeric variable antibody domain that is capable of selectivelybinding to a specific antigen (e.g. EGFR). VHH single-domain antibodiesmay be engineered from heavy-chain antibodies found in camelids. Theterms VHH or V_(H)H are used interchangeably throughout and are usedaccording to their common meaning in the art. An “EGFR VHH” or “a EGFRVHH protein” as provided herein refers to a VHH single domain antibodyspecifically binding to EGFR. In some embodiments, the EGFR VHH has thesequence set forth in SEQ ID NO: 4. In further embodiments, EGFR VHH isoperably linked to FKBP to form a ligand-dimerizing agent binderconjugate. In some further embodiments, the ligand-dimerizing agentbinder conjugate has the sequence set forth in SEQ ID NO: 6.

“CEA” or CEACAM5” as provided herein refers to carcinoembryonicantigen-related cell adhesion molecule 5 also known in the art asCD66.“CEA VHH” or “a CEA VHH protein” as provided herein refers to a VHHsingle domain antibody specifically binding to CEA. In some embodiments,the CEA VHH has the sequence set forth in SEQ ID NO: 1. In furtherembodiments, the CEA VHH is operably linked to FKBP to form aligand-dimerizing agent binder conjugate. In some further embodiments,the ligand-dimerizing agent binder conjugate has the amino acid sequenceset forth in SEQ ID NO: 3.

A “protective antigen domain 4 (D4) protein” provided herein refers tothe Bacillus anthracis protective antigen domain 4 as set forth in SEQID NO: 94. In some embodiments, D4 is operably linked to FKBP to form aligand-dimerizing agent binder conjugate. In some further embodiments,the ligand-dimerizing agent binder conjugate has the amino acid sequenceset forth in SEQ ID NO: 9.

A “control” sample or value refers to a sample that serves as areference, usually a known reference, for comparison to a test sample.For example, a test sample can be taken from a test condition, e.g., inthe presence of a test compound, and compared to samples from knownconditions, e.g., in the absence of the test compound (negativecontrol), or in the presence of a known compound (positive control). Acontrol can also represent an average value gathered from a number oftests or results. One of skill in the art will recognize that controlscan be designed for assessment of any number of parameters. For example,a control can be devised to compare therapeutic benefit based onpharmacological data (e.g., half-life) or therapeutic measures (e.g.,comparison of side effects). One of skill in the art will understandwhich controls are valuable in a given situation and be able to analyzedata based on comparisons to control values. Controls are also valuablefor determining the significance of data. For example, if values for agiven parameter are widely variant in controls, variation in testsamples will not be considered as significant.

As used herein, the term “cancer” refers to all types of cancer,neoplasm, or malignant tumors found in mammals, including leukemia,carcinomas and sarcomas. Exemplary cancers include cancer of the brain,breast, cervix, colon, head & neck, liver, kidney, lung, non-small celllung, melanoma, mesothelioma, ovary, sarcoma, stomach, uterus andMedulloblastoma. Additional examples include, Hodgkin's Disease,Non-Hodgkin's Lymphoma, multiple myeloma, neuroblastoma, ovarian cancer,rhabdomyosarcoma, primary thrombocytosis, primary macroglobulinemia,primary brain tumors, cancer, malignant pancreatic insulanoma, malignantcarcinoid, urinary bladder cancer, premalignant skin lesions, testicularcancer, lymphomas, thyroid cancer, neuroblastoma, esophageal cancer,genitourinary tract cancer, malignant hypercalcemia, endometrial cancer,adrenal cortical cancer, neoplasms of the endocrine and exocrinepancreas, and prostate cancer.

The term “leukemia” refers broadly to progressive, malignant diseases ofthe blood-forming organs and is generally characterized by a distortedproliferation and development of leukocytes and their precursors in theblood and bone marrow. Leukemia is generally clinically classified onthe basis of (1) the duration and character of the disease-acute orchronic; (2) the type of cell involved; myeloid (myelogenous), lymphoid(lymphogenous), or monocytic; and (3) the increase or non-increase inthe number abnormal cells in the blood-leukemic or aleukemic(subleukemic). The P388 leukemia model is widely accepted as beingpredictive of in vivo anti-leukemic activity. It is believed that acompound that tests positive in the P388 assay will generally exhibitsome level of anti-leukemic activity in vivo regardless of the type ofleukemia being treated. Accordingly, the present invention includes amethod of treating leukemia, and, preferably, a method of treating acutenonlymphocytic leukemia, chronic lymphocytic leukemia, acutegranulocytic leukemia, chronic granulocytic leukemia, acutepromyelocytic leukemia, adult T-cell leukemia, aleukemic leukemia, aleukocythemic leukemia, basophylic leukemia, blast cell leukemia, bovineleukemia, chronic myelocytic leukemia, leukemia cutis, embryonalleukemia, eosinophilic leukemia, Gross' leukemia, hairy-cell leukemia,hemoblastic leukemia, hemocytoblastic leukemia, histiocytic leukemia,stem cell leukemia, acute monocytic leukemia, leukopenic leukemia,lymphatic leukemia, lymphoblastic leukemia, lymphocytic leukemia,lymphogenous leukemia, lymphoid leukemia, lymphosarcoma cell leukemia,mast cell leukemia, megakaryocytic leukemia, micromyeloblastic leukemia,monocytic leukemia, myeloblastic leukemia, myelocytic leukemia, myeloidgranulocytic leukemia, myelomonocytic leukemia, Naegeli leukemia, plasmacell leukemia, multiple myeloma, plasmacytic leukemia, promyelocyticleukemia, Rieder cell leukemia, Schilling's leukemia, stem cellleukemia, subleukemic leukemia, and undifferentiated cell leukemia.

The term “sarcoma” generally refers to a tumor which is made up of asubstance like the embryonic connective tissue and is generally composedof closely packed cells embedded in a fibrillar or homogeneoussubstance. Sarcomas which can be treated with a combination ofantineoplastic thiol-binding mitochondrial oxidant and an anticanceragent include a chondrosarcoma, fibrosarcoma, lymphosarcoma,melanosarcoma, myxosarcoma, osteosarcoma, Abemethy's sarcoma, adiposesarcoma, liposarcoma, alveolar soft part sarcoma, ameloblastic sarcoma,botryoid sarcoma, chloroma sarcoma, chorio carcinoma, embryonal sarcoma,Wilms' tumor sarcoma, endometrial sarcoma, stromal sarcoma, Ewing'ssarcoma, fascial sarcoma, fibroblastic sarcoma, giant cell sarcoma,granulocytic sarcoma, Hodgkin's sarcoma, idiopathic multiple pigmentedhemorrhagic sarcoma, immunoblastic sarcoma of B cells, lymphoma,immunoblastic sarcoma of T-cells, Jensen's sarcoma, Kaposi's sarcoma,Kupffer cell sarcoma, angiosarcoma, leukosarcoma, malignant mesenchymomasarcoma, parosteal sarcoma, reticulocytic sarcoma, Rous sarcoma,serocystic sarcoma, synovial sarcoma, and telangiectaltic sarcoma.

The term “melanoma” is taken to mean a tumor arising from themelanocytic system of the skin and other organs. Melanomas which can betreated with a combination of antineoplastic thiol-binding mitochondrialoxidant and an anticancer agent include, for example, acral-lentiginousmelanoma, amelanotic melanoma, benign juvenile melanoma, Cloudman'smelanoma, S91 melanoma, Harding-Passey melanoma, juvenile melanoma,lentigo maligna melanoma, malignant melanoma, nodular melanoma, subungalmelanoma, and superficial spreading melanoma.

The term “carcinoma” refers to a malignant new growth made up ofepithelial cells tending to infiltrate the surrounding tissues and giverise to metastases. Exemplary carcinomas which can be treated with acombination of antineoplastic thiol-binding mitochondrial oxidant and ananticancer agent include, for example, acinar carcinoma, acinouscarcinoma, adenocystic carcinoma, adenoid cystic carcinoma, carcinomaadenomatosum, carcinoma of adrenal cortex, alveolar carcinoma, alveolarcell carcinoma, basal cell carcinoma, carcinoma basocellulare, basaloidcarcinoma, basosquamous cell carcinoma, bronchioalveolar carcinoma,bronchiolar carcinoma, bronchogenic carcinoma, cerebriform carcinoma,cholangiocellular carcinoma, chorionic carcinoma, colloid carcinoma,comedo carcinoma, corpus carcinoma, cribriform carcinoma, carcinoma encuirasse, carcinoma cutaneum, cylindrical carcinoma, cylindrical cellcarcinoma, duct carcinoma, carcinoma durum, embryonal carcinoma,encephaloid carcinoma, epiermoid carcinoma, carcinoma epithelialeadenoides, exophytic carcinoma, carcinoma ex ulcere, carcinoma fibrosum,gelatiniforni carcinoma, gelatinous carcinoma, giant cell carcinoma,carcinoma gigantocellulare, glandular carcinoma, granulosa cellcarcinoma, hair-matrix carcinoma, hematoid carcinoma, hepatocellularcarcinoma, Hurthle cell carcinoma, hyaline carcinoma, hypemephroidcarcinoma, infantile embryonal carcinoma, carcinoma in situ,intraepidermal carcinoma, intraepithelial carcinoma, Krompecher'scarcinoma, Kulchitzky-cell carcinoma, large-cell carcinoma, lenticularcarcinoma, carcinoma lenticulare, lipomatous carcinoma, lymphoepithelialcarcinoma, carcinoma medullare, medullary carcinoma, melanoticcarcinoma, carcinoma molle, mucinous carcinoma, carcinoma muciparum,carcinoma mucocellulare, mucoepidermoid carcinoma, carcinoma mucosum,mucous carcinoma, carcinoma myxomatodes, nasopharyngeal carcinoma, oatcell carcinoma, carcinoma ossificans, osteoid carcinoma, papillarycarcinoma, periportal carcinoma, preinvasive carcinoma, prickle cellcarcinoma, pultaceous carcinoma, renal cell carcinoma of kidney, reservecell carcinoma, carcinoma sarcomatodes, schneiderian carcinoma,scirrhous carcinoma, carcinoma scroti, signet-ring cell carcinoma,carcinoma simplex, small-cell carcinoma, solanoid carcinoma, spheroidalcell carcinoma, spindle cell carcinoma, carcinoma spongiosum, squamouscarcinoma, squamous cell carcinoma, string carcinoma, carcinomatelangiectaticum, carcinoma telangiectodes, transitional cell carcinoma,carcinoma tuberosum, tuberous carcinoma, verrucous carcinoma, andcarcinoma villosum.

By “therapeutically effective dose or amount” herein is meant a dosethat produces effects for which it is administered. The exact dose andformulation will depend on the purpose of the treatment, and will beascertainable by one skilled in the art using known techniques (see,e.g., Lieberman, Pharmaceutical Dosage Forms (vols. 1-3, 1992); Lloyd,The Art, Science and Technology of Pharmaceutical Compounding (1999);Remington: The Science and Practice of Pharmacy, 20th Edition, Gennaro,Editor (2003), and Pickar, Dosage Calculations (1999)).

The term “pharmaceutically acceptable salts” or “pharmaceuticallyacceptable carrier” is meant to include salts of the active compoundswhich are prepared with relatively nontoxic acids or bases, depending onthe particular substituents found on the compounds described herein.When compounds of the present invention contain relatively acidicfunctionalities, base addition salts can be obtained by contacting theneutral form of such compounds with a sufficient amount of the desiredbase, either neat or in a suitable inert solvent. Examples ofpharmaceutically acceptable base addition salts include sodium,potassium, calcium, ammonium, organic amino, or magnesium salt, or asimilar salt. When compounds of the present invention contain relativelybasic functionalities, acid addition salts can be obtained by contactingthe neutral form of such compounds with a sufficient amount of thedesired acid, either neat or in a suitable inert solvent. Examples ofpharmaceutically acceptable acid addition salts include those derivedfrom inorganic acids like hydrochloric, hydrobromic, nitric, carbonic,monohydrogencarbonic, phosphoric, monohydrogenphosphoric,dihydrogenphosphoric, sulfuric, monohydrogensulfuric, hydriodic, orphosphorous acids and the like, as well as the salts derived fromrelatively nontoxic organic acids like acetic, propionic, isobutyric,maleic, malonic, benzoic, succinic, suberic, fumaric, lactic, mandelic,phthalic, benzenesulfonic, p-tolylsulfonic, citric, tartaric,methanesulfonic, and the like. Also included are salts of amino acidssuch as arginate and the like, and salts of organic acids likeglucuronic or galactunoric acids and the like (see, e.g., Berge et al.,Journal of Pharmaceutical Science 66:1-19 (1977)). Certain specificcompounds of the present invention contain both basic and acidicfunctionalities that allow the compounds to be converted into eitherbase or acid addition salts. Other pharmaceutically acceptable carriersknown to those of skill in the art are suitable for the presentinvention.

A “subject,” “individual,” or “patient,” is used interchangeably herein,which refers to a vertebrate, preferably a mammal, more preferably ahuman. Mammals include, but are not limited to, murine, simian, human,farm animals, sport animals, and pets. Tissues, cells and their progenyof a biological entity obtained in vitro or cultured in vitro are alsoencompassed.

II. Compositions

Provided herein, inter alia, are adenoviral compositions useful forinfecting a broad variety of different cell types (e.g. cancer cells).For example, the compositions provided herein may be used to retargetadenovirus infection to receptors upregulated on tumor cells (e.g. EGFR,CEA, ErbB). Using the compositions provided herein including embodimentsthereof, the heterogeneity of tumors can be overcome by designingrecombinant adenoviruses that are able to infect tumor cells throughmore than one receptor. The viral compositions provided herein expresspolypeptide binding pairs (as listed in Table 2, e.g. FKBP and FRB)capable of dimerizing in the presence of a chemical dimerizing agent(e.g. rapamycin) and thereby forming a ternary complex. The ternarycomplex enables the virus to bind to a specific cellular surfacereceptor. The components of the ternary complex may completely orpartially be encoded by the adenoviral genome and are therefore not lostduring viral replication providing for the ability of the virus ofsubsequent re-infection. Thus, in one aspect, a recombinant nucleic acidencoding a capsid-dimerizing agent binder conjugate and aligand-dimerizing agent binder conjugate are provided. Thecapsid-dimerizing agent binder conjugate includes a dimerizing agentbinder (e.g. FRB) operably linked to a viral capsid protein (e.g.fiber). A dimerizing agent binder as provided herein is an agent capableof binding a dimerizing agent. A dimerizing agent binder includeswithout limitation a protein, a compound or a small molecule. In someembodiments, the dimerizing agent binder is a FRB protein. Non limitingexamples of dimerizing agent binders are set forth in Table 2 providedherein. Binding of the dimerizing agent binder to the dimerizing agentmay occur through non-covalent intermolecular interactions such ashydrogen bonding, electrostatic interactions, hydrophobic and Van derWaals forces. The capsid-dimerizing agent binder conjugate includes aviral capsid protein. The term capsid refers to any component (e.g.capsid proteins or polypeptides) forming the shell of a virus, whereinthe capsid can include one or more of these components. The capsidincludes any appropriate structural components of the viral shell. Insome embodiments, the capsid protein is an adenoviral capsid protein.Non-limiting examples of capsid proteins are L3 II (hexon) (e.g.encoding major structural proteins that form the triangular faces of thecapsid), L1 Ma (e.g. encoding minor structural proteins that help tostabilize the capsid), L2 III (penton) (e.g. encoding major structuralproteins that form the vertex of the capsid where the fiber protrudes),L2 pVII (e.g. encoding core structural proteins with homology to histoneH3 and associate with viral DNA in the capsid), and L5 IV (Fiber) (e.g.encoding major structural proteins that extend from the penton base andare responsible for receptor binding). In some embodiments, theadenoviral capsid protein is a fiber protein.

Upon expression in a cell the dimerizing agent binder and the viralcapsid protein form a capsid-dimerizing agent binder conjugate, which iscapable of binding to a dimerizing agent (e.g. rapamycin) through thedimerizing agent binder (e.g. FRB) and is incorporated into the viralcapsid by the capsid protein (e.g. fiber). Thus, in some embodiments,the capsid-dimerizing agent binder conjugate includes a capsid proteinand a dimerizing agent binder. In other embodiments, the capsid proteinis operably linked to the dimerizing agent binder. Through binding tothe dimerizing agent the capsid-dimerizing agent binder conjugate mayconnect to the ligand-dimerizing agent binder conjugate. Theligand-dimerizing agent binder conjugate includes a cell surfacereceptor-specific ligand (e.g. EGFR VHH) operably linked to a seconddimerizing agent binder (e.g. FKBP). A ligand as provided herein is aprotein with the capability of binding a molecule expressed on thesurface of a cell. Non-limiting examples of ligands and correspondingcellular receptors are set forth in Table 3. In some embodiments, theligand is a EGFR VHH protein. In a further embodiment, the dimerizingagent binder is FKBP. In some embodiments, the ligand is a CEA VHHprotein. In a further embodiment, the dimerizing agent binder is FKBP.In some embodiments, the ligand is a protective antigen domain 4 (D4)protein. In a further embodiment, the dimerizing agent binder is FKBP.In some embodiments, the ligand-dimerizing agent binder conjugateincludes a ligand and a dimerizing agent binder. In some embodiments,the ligand is operably linked to the dimerizing agent binder. In someembodiments, the ligand is an antibody. In some further embodiments, theantibody is a single domain antibody. In some embodiments, a pluralityof ligands is operably linked to the dimerizing agent binder, whereinthe plurality of ligands are individually different. The plurality ofligands may be operably linked to one or both termini of the dimerizingagent binder. In some embodiments, the plurality of ligands is operablylinked in tandem to one or both termini of the dimerizing agent binder.In some embodiments, the dimerizing agent binder is an immunophilinprotein. In some further embodiments, the immunophilin protein is a FKBPprotein. In some further embodiments, the FKBP protein is a human FKBPprotein. In some further embodiments, the human FKBP protein is FKBP12.In other embodiments, the ligand is capable of binding a cell. In otherembodiments, the cell is a tumor cell.

Provided herein, inter alia, are recombinant adenoviruses expressing therecombinant nucleic acid described above. Thus, in another aspect, arecombinant adenovirus including a recombinant nucleic acid providedherein including embodiments thereof is provided. In some embodiments,the adenovirus is a replication incompetent adenovirus. In otherembodiments, the adenovirus is a replication competent adenovirus. Wherethe adenovirus is a replication competent adenovirus, the adenovirus iscapable of infecting a cell by binding to a specific cellular surfacereceptor (e.g. EGFR), replicating inside said cell thereby producing newviral progeny capable of infecting additional cells. In contrast, areplication incompetent adenovirus, is capable of entering a cell bybinding to a specific cellular receptor and expressing the adenoviralgenome inside said cell. However, a replication incompetent virus lacksgenes necessary to produce new viral progeny and therefore is notcapable of subsequent infection of additional cells.

In another aspect, a recombinant adenovirus including acapsid-dimerizing agent binder conjugate is provided. As described abovea capsid-dimerizing agent binder conjugate includes a capsid protein(e.g. fiber) operably linked to a dimerizing agent binder (e.g. FRB).The binding of the dimerizing agent binder (e.g. FRB) to the dimerizingagent (e.g. rapamycin) therefore connects the recombinant adenovirus tothe dimerizing agent. Thus, the recombinant adenovirus including acapsid-dimerizing agent binder conjugate may be bound to a dimerizingagent. A dimerizing agent as provided herein is an agent capable ofbinding a dimerizing agent binder of a capsid-dimerizing agent binderconjugate and a dimerizing agent binder of a ligand-dimerizing agentbinder conjugate. In some embodiments, the dimerizing agent binds adimerizing agent binder of a capsid-dimerizing agent binder conjugate.The dimerizing agent may bind a dimerizing agent binder of aligand-dimerizing agent binder conjugate and a dimerizing agent binderof a ligand-dimerizing agent binder conjugate. Thus, in someembodiments, the dimerizing agent is further bound to aligand-dimerizing agent binder conjugate. A dimerizing agent may bindthe dimerizing agent binder through non-covalent intermolecularinteractions such as hydrogen bonding, electrostatic interactions,hydrophobic and Van der Waals forces. In some embodiments, thedimerizing agent binds covalently to the dimerizing agent binder. Thedimerizing agent as provided herein may be a naturally occurringsubstance (e.g. rapamycin, abscisic acid) or a synthetic substance (e.g.a small molecule, a compound). Examples of dimerizing agents accordingto the invention provided herein are listed in Table 2. In someembodiments, the dimerizing agent is a compound. In some furtherembodiments, the compound is rapamycin. Rapamycin refers, in thecustomary sense, to CAS Registry No. 53123-88-9. Rapamycin inhibits themTOR kinase and is used as an immunosuppressing agent and anti-cancertreatment. In some further embodiments, the dimerizing agent is arapalog. A rapalog as provided herein is a rapamycin analog does notinhibit cellular mTOR kinase activity. In some further embodiment, therapalog is AP21967. In some embodiments, the dimerizing agent is ananti-cancer drug.

As described above, the compositions provided herein include arecombinant adenovirus including a recombinant nucleic acid including acapsid-dimerizing agent binder and a ligand-dimerizing agent binderconjugate. Therefore, the recombinant adenovirus may further include aligand-dimerizing agent binder conjugate. In other embodiments, theligand-dimerizing agent binder conjugate is ectopically expressed.Wherein the ligand-dimerizing agent binder conjugate is ectopicallyexpressed, the nucleic acid encoding the ligand-dimerizing agent binderconjugate does not form part of the recombinant nucleic acid included inthe recombinant adenovirus. Where the ligand-dimerizing agent binderconjugate is ectopically expressed it may be encoded by the genome ofthe cell infected with the recombinant adenovirus.

In some embodiments, the recombinant adenovirus includes a plurality ofligand-dimerizing agent binder conjugates, wherein eachligand-dimerizing agent binder conjugate may be different. For examplewhere the recombinant adenovirus includes a plurality ofligand-dimerizing agent binder conjugates, the recombinant adenovirusmay include a first ligand-dimerizing agent binder conjugate, a secondligand-dimerizing agent binder conjugate and a third ligand-dimerizingagent binder conjugate with each ligand-dimerizing agent binderconjugate being different. Thus, the first ligand-dimerizing agentbinder conjugate may include a first ligand and a first dimerizing agentbinder, the second ligand-dimerizing agent binder conjugate may includea second ligand and a second dimerizing agent binder, wherein the firstligand is different from the second ligand and the first dimerizingagent binder is the same or different from the second dimerizing agentbinder. For example, the first ligand-EGFR VHH may be operably linked tothe first dimerizing agent binder FKBP and the second ligand CEA VHH maybe operably linked to the second dimerizing agent binder AB1 or FKBP.

Moreover, the recombinant adenovirus may include a plurality ofcapsid-dimerizing agent binder conjugates, wherein eachcapsid-dimerizing agent binder conjugate may be different. For examplewhere the recombinant adenovirus includes a plurality ofcapsid-dimerizing agent binder conjugates, the recombinant adenovirusmay include a first capsid-dimerizing agent binder conjugate, a secondcapsid-dimerizing agent binder conjugate and a third capsid-dimerizingagent binder conjugate with each capsid-dimerizing agent binderconjugate being different. Thus, the first capsid-dimerizing agentbinder conjugate may include a first capsid protein and a firstdimerizing agent binder, the second capsid-dimerizing agent binderconjugate may include a second capsid protein and a second dimerizingagent binder, wherein the first and second capsid protein may be thesame or different and the first and second dimerizing agent binder maythe same or different. For example, the first capsid protein fiber maybe operably linked to the first dimerizing agent binder FRB and thesecond capsid protein fiber may be operably linked to the seconddimerizing agent binder PYL1. Thus, in one embodiment, the recombinantadenovirus includes a first capsid-dimerizing agent binder conjugate(e.g. fiber/FRB), a first ligand-dimerizing agent binder conjugate (e.g.EGFR VHH/FKBP), a second capsid-dimerizing agent binder conjugate (e.g.fiber/PYL1) and a second ligand-dimerizing agent binder conjugate (e.g.CEA VHH/AB1). In the presence of a first dimerizing agent (i.e.rapamycin) the first capsid-dimerizing agent binder conjugate and thefirst ligand-dimerizing agent binder conjugate are connected through thebinding of FRB and FKBP to rapamycin. In the presence of a seconddimerizing agent (i.e. abscisic acid) the second capsid-dimerizing agentbinder conjugate and the second ligand-dimerizing agent binder conjugateare connected through the binding of AB1 and Pyl1 to abscisic acid.Therefore, in the presence of rapamycin the recombinant adenovirusinfects cells expressing the EGF receptor and in the presence ofabscisic acid the same virus may infect cells expressing CEA. Thus, thesame recombinant adenovirus is capable of infecting different cell typesdepending on the presence of dimerizing agent administered.

In another aspect, a cell including a recombinant adenovirus providedherein including embodiments thereof is provided. In some embodiments,the cell is a cancer cell in a cancer patient. In other embodiments, thecell is a non-cancer cell in a cancer patient. In some embodiments, thecell is a cell in an organism. In some further embodiments, the organismis a mammal. In some further embodiments, the mammal is a human. Inother embodiments, the cell is a cell in a culture vessel. In somefurther embodiments, the cell is a transformed cell.

III. Methods

In another aspect, a method of forming an adenoviral cancer celltargeting construct is provided. The method includes infecting a cellwith a recombinant adenovirus provided herein, thereby forming anadenoviral infected cell. The adenoviral infected cell is allowed toexpress the recombinant nucleic acid, thereby forming aligand-dimerizing agent binder conjugate and a recombinant adenovirusincluding a capsid-dimerizing agent binder conjugate. The recombinantadenovirus and the ligand-dimerizing agent binder conjugate arecontacted with a dimerizing agent. The recombinant adenovirus and theligand-dimerizing agent binder conjugate are allowed to bind to thedimerizing agent, thereby forming the adenoviral cancer cell targetingconstruct. As described above, the recombinant nucleic acid may includea plurality of capsid-dimerizing agent binder conjugates andligand-dimerizing agent binder conjugates, thereby enabling theadenovirus expressing the recombinant nucleic acid to bind to pluralityof different cellular surface receptors.

In another aspect, a method of targeting a cell is provided. The methodincludes contacting a cell with a recombinant adenovirus provided hereinincluding embodiments thereof. In some embodiments, the cell is a cancercell.

In another aspect, a method of targeting a cancer cell in a cancerpatient is provided. The method includes administering to a cancerpatient a recombinant adenovirus provided herein. The recombinantadenovirus is allowed to infect a cell in the cancer patient, therebyforming an adenoviral infected cell. The adenoviral infected cell isallowed to express the recombinant nucleic acid, thereby forming aligand-dimerizing agent binder conjugate and a recombinant adenovirusincluding a capsid-dimerizing agent binder conjugate. The cancer patientis administered with a dimerizing agent. The recombinant adenovirus andthe ligand-dimerizing agent binder conjugate are allowed to bind to thedimerizing agent, thereby forming an adenoviral cancer cell targetingconstruct. The adenoviral cancer cell targeting construct is allowed tobind to a cancer cell, thereby targeting the cancer cell in the cancerpatient. In some embodiments, the cell is a cancer cell. In otherembodiments, the cell is a non-cancer cell.

In another aspect, a method of targeting a cell is provided. The methodincludes contacting a first cell with a recombinant adenovirus providedherein. The recombinant adenovirus is allowed to infect the first cell,thereby forming an adenoviral infected cell. The adenoviral infectedcell is allowed to express the recombinant nucleic acid, thereby forminga ligand-dimerizing agent binder conjugate and a recombinant adenoviruscomprising a capsid-dimerizing agent binder conjugate. Theligand-dimerizing agent binder conjugate and the recombinant adenovirusare contacted with a dimerizing agent. The recombinant adenovirus andthe ligand-dimerizing agent binder conjugate are allowed to bind to thedimerizing agent, thereby forming an adenoviral cell targetingconstruct. The adenoviral cell targeting construct is allowed to bind toa second cell, thereby targeting said cell. In some embodiments, thefirst cell and the second cell form part of an organism.

IV. Specific Embodiments

Cancer is a debilitating disease that accounts for more than half amillion deaths each year. There is a profound need for more effective,selective and safe treatments for cancer. Existing treatments for thispervasive, life threatening disease, such as chemotherapy and surgery,rarely eliminate all malignant cells, and often exhibit deleteriousside-effects that can outweigh therapeutic benefit. The presentinvention provides powerful recombinant viruses that are capable ofinfecting tumor cells via disparate receptors. These viruses will enablea new safe form of effective, self-amplifying therapy that breaks theparadigm of systemic genotoxic treatments for cancer.

One approach that has the potential to address many of the shortcomingsof current cancer treatments is oncolytic adenoviral therapy (Pesonen,S. et al., Molecular Pharmaceutics, 8(1): p. 12-28 (2010)). Theseviruses are designed to replicate specifically in cancer cells, butleave normal cells unharmed. This selectivity can be engineered byexploiting the functional overlap between adenoviral, earlyonco-proteins, such as E1A, and tumor mutations in the Rb tumorsuppressor pathway which drives deregulated cell cycle entry andpathological DNA replication (Poznic, M., J Biosci, 34(2): p. 305-12(2009)).

Adenovirus (Ad) is a self-replicating biological machine. It consists ofa linear double-stranded 36 kb DNA genome sheathed in a protein coat. Adrequires a human host cell to replicate. It invades and hijacks thecellular replicative machinery to reproduce and upon assembly induceslytic cell death to escape the cell and spread and invade surroundingcells (FIG. 1). No ab initio system has come close to mimicking theautonomy and efficiency of Ad, however, Applicants have developed twonew strategies to systematically manipulate the Ad genome to createnovel adenoviruses as described in published applicationPCT/US2011/048006, which is herein incorporated in its entirety and forall purposes. Henceforth, with the ability to manipulate the Ad genome,Applicants can take the virus by the horns and redesign it to performthe functions of tumor-specific infection, replication, and cellkilling.

Currently, adenoviral vectors rely on a single cellular receptor fortheir uptake, which significantly limits their therapeutic potential.Ad5 infection is mediated primarily through interactions between thefiber protein on the outer viral capsid and the coxsackie and adenovirusreceptor (CAR) on human epithelial cells. Unfortunately, many cancercells do not express CAR, such as mesenchymal and deadly metastatictumor cells. Since viral replication/killing will be limited by theability to infect cells, Applicants need viruses that infect tumor cellsvia receptors other than CAR, ideally those specifically upregulated ontumor cells. Provided herein are genetically-encoded switchabletargeting moieties that enable Ad5 to infect cancer cells regardless oftheir CAR-expression. Applicants used a known property of the cancerdrug rapamycin (rap) to dimerize heterologous proteins with FKBP and FRBdomains and engineered viruses that express a FRB-fiber capsid proteinfusion together with retargeting ligands fused to FKBP. These virusesare induced to infect any cell type via multiple retargeting ligandsupon rap treatment. This represents a rational and powerful combinationof chemical and viral weapons as a novel cancer therapy. In addition, amajor goal is to overcome tumor heterogeneity by engineering virusesthat are able to infect tumor cells through more than one more mechanismand receptor. Applicants achieved this by using a known property of thecancer drug rapamycin (rap) to dimerize heterologous proteins with FKBPand FRB domains. Rapamycin inhibits the mTOR kinase and is used as animmunosuppressing agent and anti-cancer treatment. By engineering FRBmutations, rapalogs of rapamycin that do not inhibit cellular mTORkinase activity can also be used to induce infection of any cell typeupon administration of a rapalog. These viruses can be induced to infectany cell type via multiple retargeting ligands upon rap treatment.

Cancer continues to be an intractable disease without safe and reliablyeffective treatments. In the last century, Applicants' knowledge aboutthe origins of cancer and cancer biology has greatly advanced. However,despite Applicants' new understanding of cancer as a genetic disease,the standard of care for non-resectable disseminated disease remainsgenotoxic therapies, such as chemotherapy and irradiation, which oftenhave intolerable and toxic side-effects. While drugs have been developedto target oncogenic proteins, Applicants have nothing to treat thegenetic loss of tumor-suppressors. One approach to treat these cancersis oncolytic viral cancer therapy (FIG. 1) (Pesonen, S. et al.,Molecular Pharmaceutics, 8(1): p. 12-28 (2010)).

Adenovirus (Ad) has been studied for more than half a century, and hascontributed significantly to Applicants' understanding of key mechanismsat the heart of mammalian cell biology such as splicing, critical growthregulatory hubs, transcription, and the cell cycle. Ad is a smalldouble-stranded 36 kb DNA virus, sheathed in a protein capsid coat (FIG.2). Ad particles primarily interact with host cells through proteininteractions between the knob-domain of fiber on the surface of thecapsid and a cell surface molecule (FIG. 2). Serotype 5 of adenovirusspecies C (Ad5) infects cells via fiber interactions with coxsackievirusand adenovirus receptor (CAR), primarily found at epithelial celljunctions. Like all viruses, it is entirely dependent on host cells forits propagation. After depositing its genome into the host cell nucleus,a program is coordinated by virus proteins to activate the cell cycle inquiescent cells in order to replicate virus DNA. At the end of the Ad5life cycle, after progeny virions have been assembled in the cellnucleus, the membranes of the cell are lysed, releasing the nextgeneration of viruses.

Manipulating Adenovirus Tropism

Ad5 infection is mostly limited to cells that have CAR, which isexpressed along with cadherin at epithelial cell tight junctions (Tomko,R. P. et al., Proceedings of the National Academy of Sciences, 94(7): p.3352-3356 (2009); Bergelson, J. M., et al., Science, 275(5304): p.1320-1323 (1997)). Unfortunately, it is metastases that kill most cancerpatients, in which an epithelial to mesenchymal transition (EMT) resultsin downregulation of cadherin and CAR, instigating invasion and spreadto distant sites (Anders, M., et al., Br J Cancer, 100(2): p. 352-9(2009)). Thus, many malignant cells do not express CAR and are notsusceptible to infection by Ad5 (Anders, M., et al., Br J Cancer,100(2): p. 352-9 (2009); Dietel, M., et al., Journal of MolecularMedicine, 89(6): p. 621-630 (2011); Matsumoto, K., et al., Urology,66(2): p. 441-446 (2005)). A number of approaches have been taken toretarget Ad5 to different cellular receptors, including: chemicalmodification of purified adenovirus particles and infection withrecombinant divalent “bridging” proteins to form complexes between fiberand receptor (reviewed in (Rein, D. T., M. Breidenbach, and D. T.Curiel, Future Oncology, 2(1): p. 137-143 (2006))). The disadvantage ofthese approaches is the restriction to the first round of infection,since following virus replication the chemical/recombinant targetingmoiety is lost. This drawback can be overcome by directly modifying thefiber gene to encode targeting sequences, however this approach is notsystematic because Applicants cannot predict the folding of de novosequences and the correct assembly with virus particles. To date, thisapproach has only been useful for the insertion of small peptides.

Adsembly and AdSLIC are Enabling Technologies to Systematically DesignNew Optimized Adenoviruses From Libraries of Genomic Building Blocks andHeterologous Parts

The potential of adenoviral vectors in several applications is hinderedby the ability to engineer and combine multiple genetic modificationsrapidly and systematically. To systematically re-design adenovirus as anoncolytic agent, Tools are needed to enable precise modification of itscomponents. The 36 kb Ad5 genome is difficult to manipulate due to itssize and abundance of restriction enzyme recognition (RER) sites. Todate, a majority of recombinant Ads have been limited to the backbonesthat were digested and selected for fewer RER sites in the 1980s, andcontinue to remain due to the legacy of shuttle vectors. These backboneshave accumulated a number of mutations distant from wild type sequences.Traditional cloning techniques with complex sequences are still timeconsuming and not systematic.

To overcome the limitations of Ad5 and current methodologies, Applicantshave developed two new technologies, named ‘Adsembly’ and ‘AdSlicR’,which enable the rapid de novo assembly of adenoviral genomes in vitrofrom genomic component parts and heterologous elements in a single hour.Using a bioinformatics approach, Applicants split the adenoviral genome(36 kb) into 5 units, based on evolutionarily conserved sequencesbetween species, transcriptional and functional modules. Each of these 5units comprise compatible sections of a genomic building “partslibrary”, the functions and diversity of which can be altered byengineering mutations or heterologous elements and further expanded byadding equivalent units from disparate adenovirus serotypes, mutants andspecies. In order to create a new adenovirus with unique properties, oneof each of the units is selected from the library and rapidlyreassembled into a complete genome in vitro using Adsembly or Ad-SlicR.Adsembly can be used to assemble a novel genome (in 1 hour) viamulti-site specific recombination, which upon transfection, self-excisesfrom a plasmid backbone and replicates to produce novel viruses.Ad-SlicR, which utilizes the same library genome building blocks, is acomplementary strategy to erase inserted recombination sequences formore potent viral replication (if necessary) and clinical use. The easeof manipulation of multiple genomic fragments as small modular plasmidunits and the systematic approach of these technologies now allows forrapid and precise construction of novel adenoviruses.

Adenovirus Targeting: A Genetically Encoded Switch

Oncolytic viral therapy has the potential to destroy a tumor mass ofunlimited size, but only if the virus crosses the tumor vasculature andinfection spreads from one cell to another. The fiber of Ad5 recognizesthe epithelial cell junction molecule CAR (Tomko, R. P. et al.,Proceedings of the National Academy of Sciences, 94(7): p. 3352-3356(2009); Bergelson, J. M., et al., Science, 275(5304): p. 1320-1323(1997)), which is expressed in variable levels on tumors (Rein, D. T.,M. Breidenbach, and D. T. Curiel, Future Oncology, 2(1): p. 137-143(2006); Dmitriev, I., et al., J. Virol., 72(12): p. 9706-9713 (1998);Bauerschmitz, G. J., S. D. Barker, and A. Hemminki, Int J Oncol, 21(6):p. 1161-74 (2002); Breidenbach, M., et al., Hum Gene Ther., 15(5): p.509-18 (2004); Cripe, T. P., et al., Cancer Res, 61(7): p. 2953-60(2001); Fechner, H., et al., Gene Ther, 7(22): p. 1954-68 (2000); Hemmi,S., et al., Hum Gene Ther, 9(16): p. 2363-73 (1998); Hemminki, A. and R.D. Alvarez, BioDrugs, 16(2): p. 77-87 (2002); Kanerva, A., et al., ClinCancer Res, 8(1): p. 275-80 (2002); Li, Y., et al., Cancer Res, 59(2):p. 325-30 (1999); Miller, C. R., et al., Cancer Res, 58(24): p. 5738-48(1998); Rein, D. T., et al., Int J Cancer, 111(5): p. 698-704 (2004))and the loss of which is associated with increased metastasis (Anders,M., et al., Br J Cancer, 100(2): p. 352-9 (2009); Dietel, M., et al.,Journal of Molecular Medicine, 89(6): p. 621-630 (2011); Matsumoto, K.,et al., Urology, 66(2): p. 441-446 (2005)). Ad5 is not a naturallyblood-borne virus and does not actively target and cross thevasculature. Both of these factors limit the potential of adenoviralvectors for gene expression and therapy.

Attempts to retarget adenoviral uptake include the use of chemicaladapters that link viral capsids to retargeting ligands. One example isfiber biotinylation to provide a chemical linker for high affinitybinding to avidin-retargeting ligands (Liu, Y., P. Valadon, and J.Schnitzer, Virology Journal, 7(1): p. 316 (2010)). However, retargetingis only achieved with exogenous virus, since the chemical modificationsare lost upon viral replication. Genetically encoding retargetingadapter fusions to viral coat proteins is desirable, but also morechallenging. Unfortunately, the incorporation of large ligands in capsidproteins disrupts their folding/assembly (Belousova, N., et al., J.Virol., 76(17): p. 8621-8631 (2002)). To avoid misfolding, smallerpolypeptides can be inserted into the fiber H1 loop (FIG. 6) (Belousova,N., et al., J Virol, 76(17): p. 8621-31 (2002)). For example, RGDpeptides enhance integrin-assisted uptake, but are not sufficient toalter viral tropism. Fiber fusions to single chain antibodies (scFVs)are attractive as well, but the former require processing in theER/cytosol while fiber assembles in the nucleus (Kontermann, R. E., CurrOpin Mol Ther, 12(2): p. 176-83 (2010)). Thus, despite ongoing effortsto retarget infection, in vivo studies and gains have been disappointing(Waehler, R., S. J. Russell, and D. T. Curiel, Nat Rev Genet, 8(8): p.573-87 (2007)).

An ideal virus would cross the blood/endothelium layer and infect tumorcells via disparate receptors. The ideal system would be a geneticallyencoded chemical adapter that could be used to switch viral tropismwithin the body via any multiple retargeting moieties, withoutcompromising viral replication and safety. Provided herein is a novel,inducible, genetically encoded chemical adapter system that retargetsinfection to multiple cell types, and is not lost upon viralreplication. The present invention therefore overcomes the limitationsof prior approaches and has several advantages. Any unanticipatedtoxicities associated with receptor-retargeting can be stopped by drugwithdrawal. In addition, multiple retargeting ligands can be expressedwithin a single virus to target tumor cell receptors (e.g. EGFR) and thevasculature (e.g. Von Willebrand factor/transferrin).

Applicants retargeted the adenovirus viral coat protein fiber toalternate cellular receptors using a known property of theimmunosuppressive and anti-tumor drug rapamycin (rap; FIG. 7). Rapamycincan be used to induce heterodimers of heterologous proteins if one isfused to FKBP domains (e.g. a retargeting ligand) and the other (e.g.fiber) to the FRB domain of mTOR (Chen, J., et al., Proc Natl Acad SciUSA, 92(11): p. 4947-51 (1995)). Upon treatment with rap, fiber willheterodimerize with the retargeting ligand enabling the virus to infectthe cell type of choice. Rapamycin is a macrolide antibiotic that is FDAapproved and has ideal pharmacokinetic profiles in mammals. The highaffinity and stability of rap-induced heterodimerization has been usedwith great success in several applications including phage display ofreceptor-ligand complexes (de Wildt, R. M., et al., Proc Natl Acad SciUSA, 99(13): p. 8530-5 (2002)), transcriptional activation andreconstitution of bi-functional proteins (Clackson, T., Chem Biol DrugDes, 67(6): p. 440-2 (2006)). A novel application of this system isprovided, which also takes advantage of Applicants' previous studies ofrap as a rational combination with oncolytic viruses (O'Shea, C., etal., Embo J, 24(6): p. 1211-21 (2005)).

Develop a Genetically Encoded Small Molecule-Controlled System forRetargeting Adenovirus to Tumor Cell Receptors

The reliance of current adenoviral vectors on a single cellular receptorfor their uptake limits their therapeutic potential via systemicdelivery. To overcome this problem, Applicants designed novel Ads withthe rapamycin-induced, genetically encoded FRB/FKBP heterodimer toenable retargeting of adenovirus to tumor cell receptors. Ultimately,this system enables targeting of receptors in angiogenic tumorvasculature to eliminate aggressive tumors (e.g. TEMs, TVMs), andupregulated markers in high-risk tumors such as breast cancer (e.g.EGFR, HER2, TfR).

Insertion of FRB Domain Into Fiber H1 Loop

The fiber protein which infers tropism to adenovirus is generally notpermissible to large insertions or modifications, because the correctfolding and assembly of fiber trimers into adenovirus particles arecritical for viable progeny. To date, insertion of sequences in theC-terminal Ad5 fiber knob-domain has been effectively limited topeptides (Belousova, N., et al., J. Virol., 76(17): p. 8621-8631(2002)). Using Adsembly (described in FIG. 5) the 90 amino acid FRBdomain was inserted into the flexible H1 loop of fiber, whichaccommodates insertions of up to 100 amino acids without deleteriouseffects (Belousova, N., et al., J. Virol., 76(17): p. 8621-8631 (2002)).

Experimental Approach

The wild type E3 component plasmid, E₃₋₀₀₁, (Table 1) from the genomeparts library which Applicants designed was used as the template forinsertion of the FRB sequence into the fiber gene. E3-001 was PCRamplified to generate a product with SLIC-compatible ends for insertionbetween fiber Thr546 and Pro567. The 90 aa FRB domain of mTOR(Glu2025-Gln2114) was PCR amplified from mTOR cDNA and combined via SLICto generate E3-002 (FIG. 8). Wild-type E2, L3, and E4 components werecombined with E3-002 and E1-009 (containing a CMV-driven GFP gene) usingthe Adsembly strategy to generate the Ad-122 genome (FIG. 8). Applicantstransfected the Ad-122 genome into 293 E4 cells, and harvested virus.Unlike the insertion of many large ligands such as TfR, FRB did notinhibit viral replication or assembly and robust infection as evidencedby GFP fluorescence from the E1 reporter and observed cytopathic effect(CPE). I confirmed expression of FRB-fiber by Western blot as indicatedby predicted migration of FRB-fiber (72.4 kDa) versus wt fiber (61.6kDa; FIG. 9).

Expression of FKBP Retargeting Moieties From Adenovirus Genome

The expression of FKBP from the virus genome would ideally have similartiming and levels matching that of fiber, to enable efficientdimerization with fiber in the presence of rap. Applicants adoptedseveral strategies to express FKBP from the genome as summarized in FIG.10.

Experimental Approach

The E3-002 plasmid, carrying the FRB insertion in fiber, was used as thetemplate to introduce the sequences necessary for the strategiessummarized in FIG. 10. The first approach was to express FKBP from theadenovirus genome by co-translationally expressing it from the fibertranscript (FIG. 10C). The FKBP sequence was placed downstream of thefiber coding sequence following an inserted Furin-2a sequence. TheFurin-2a sequence is an optimized Furin protease recognition sitefollowed by the foot-and-mouth disease virus 2a auto-cleavage site(Fang, J., et al., Mol Ther, 15(6): p. 1153-1159 (2007)). It shouldgenerate two distinct polypeptides in equimolar amount; the FRB-Fibermolecule with a residual arginine on its C-terminus, and the FKBPprotein with residual proline on its N-terminus.

The sequence was cloned successfully, generating E3-016, and used theAdsembly strategy to create a full length genome with E1-009, E3-016 andwild type E2, L3, and E4. Similar to the preparation of Ad-122, theisolated supernatant was applied to 293 E4 cells, but there was noproductive viral replication indicated by either fluorescence or CPE.Therefore, an alternative approach was used to express FKBP using anIRES element inserted on the 3′ end of the fiber gene before the polyAsequence used for the fiber transcript (FIG. 10D). The E3 component(E3-015) was cloned successfully, and generated a complete genome usingE1-009, E3-015 and wildtype E2, L3, and E4 (FIG. 8). This virus was ableto replicate in 293 E4 cells, however no FKBP expression could bedetected by Western blot as late as 60 h p.i., indicating that theefficiency of an IRES on the fiber transcript is not ideal to expressFKBP. The final approach was to utilize adenovirus transcriptionalarchitecture to express FKBP. Since the genes in the E3 transcriptionunit of adenovirus are dispensable for virus replication in cellculture, the sequence on the E3B transcript encoding RIDα, RIDβ, and14.7 k was replaced with FKBP. The E3 component (E3-048) was cloned andused Adsembly with E1-009, wild type E2, L3, and E4 to generate Ad-178(FIG. 8). This virus was able to replicate in 293 E4 cells, as evidencedby fluorescence and CPE. Western blot analysis of infected cell lysatesrevealed that the FKBP protein accumulated in infected 293 E4 cells(FIG. 11). Therefore, this strategy was used to create novel virusesthat express FRB-fiber and FKBP retargeting moieties.

Targeting Moieties for Fusion to FKBP

An ideal targeting protein for fusion to FKBP is a stable molecule withstrong affinity for a specific cancer cell surface molecule. A number ofapproaches were explored including: BN peptide, EGF peptide, TGFα,anthrax toxin PA, Tf, F3 peptide, and VEGF (Table 3). As a proof ofprinciple, VHHs, were first explored as described below. A similarexperimental approach will be adapted for other retargeting moietieslisted in Table 3. A class of proteins which best fits these criteriaare the heavy chain domains (VHH) from single-domain antibodies (sdAbs).Camelids and sharks encode sdAbs which have specificity for theirspecific target from one variable chain domain, instead of the two(conventionally) that most other mammals have (e.g. rodents, humans)(Kontermann, R. E., Curr Opin Mol Ther, 12(2): p. 176-83 (2010)).Although small single-chain variable fragments (scFVs) have been morewidely used, the smaller and more stable VHHs have the distinctadvantage of not requiring post-translational disulfide bond formationto function. FKBP was fused to VHHs with specificity to cancer cellreceptors to impart ideal adenovirus targeting. To demonstrate thiseffect with the rap-inducible retargeting system, recently identifiedVHHs with specificity to carcinoembryonic antigen-related cell adhesionmolecule 5 (CEA also CEACAM5) (Vaneycken, I., et al., Journal of NuclearMedicine, 51(7): p. 1099-1106 (2010); Behar, G., et al., FEBS Journal,276(14): p. 3881-3893 (2009)), a biomarker for gastrointestinal, breast,lung and ovarian carcinomas (Duffy, M. J., Clin Chem, 47(4): p. 624-630(2001)), and epidermal growth factor receptor (EGFR) (Gainkam, L. O., etal., Journal of nuclear medicine: official publication, Society ofNuclear Medicine, 49(5): p. 788-95 (2008)), upregulated in many cancersof epithelial origin such as breast, head and neck, prostate, lung, andskin are used. Based on the structural modeling (FIG. 13), the VHHdomains (CEAVHH, EGFRVHH) are fused to the N terminus of FKBP for theleast steric hindrance for VHH/target interactions and the FKBP/rap/FRBdimerization interface.

The gene sequences encoding CEAVHH and EGFRVHH were human codonoptimized and synthesized by Blue Heron Biotechnologies based on proteinsequences identified by Behar et al. and Roovers et al., respectively(Behar, G., et al., FEBS Journal, 276(14): p. 3881-3893 (2009); Roovers,R., et al., Cancer Immunology, Immunotherapy, 56(3): p. 303-317 (2007)).Using SLIC, the VHH sequences were fused to the N-terminus of FKBP withan inserted GSGSGST linker sequence. These fusion proteins were clonedinto E3 components with the approach described in herein to generateAd-177 and Ad-178 (Table 1). FIG. 11 shows the expression theEGFRVHH-FKBP fusion protein from the Ad-178 infected cells, which issimilar to CEAVHH-FKBP expression from Ad-177 (data not shown). The genesequence encoding PA domain 4 were human codon optimized and synthesizedby Blue Heron Biotechnologies based on Uniprot accession P13423. UsingSLIC, the PA domain 4 was fused to the N-terminus of FKBP with aninserted GSGSGST linker sequence. This fusion protein was cloned into anE3 component with the approach described in herein to generate Ad-281(Table 1).

Immunofluorescence to Detect Rapamycin-Induced Colocalization ofFRB-Fiber and VHH-FKBP Fusion Proteins

Detection of the colocalization of proteins in cells byimmunofluorescence or via fluorescently tagged proteins is one approachto evaluate if proteins have the potential for interaction. A differenceof FKBP localization to FRB-fiber (or vice versa) in the presence of rapversus the absence would suggest that rap was inducing theirassociation. 293 E4 cells grown on microscope slides for direct imagingof adenovirus expressed proteins are infected. FRB-fiber and VHH-FKBPfusion proteins are evaluated in cells which have 500 nM rap versussolvent control to evaluate any differences in localization due topresence of the drug. As controls, a virus with the Adsembly strategy isconstructed that expresses CEAVHH-FKBP and wt fiber (Ad-199) as acontrol for FRB-dependent rap-induced colocalization of FKBP.Non-confocal IF imaging in infected 293 E4 cells shows colocalization ofFRB-fiber and VHH-FKBP signals in the presence of rap (FIG. 14).

Co-Immunoprecipitation (CoIP) of FRB-Fiber and VHH-FKBP Fusion ProteinsVia Rapamycin Induced Heterodimerization

CoIP of FRB-fiber through IP of FKBP (and vice versa) from the lysatesof infected 293 E4 cells have been performed. Viruses used are theexperimental group, Ad-177 or Ad-178, to evaluate rap-induced FRB-FKBPassociation; Ad-122 (no FKBP, FRB-fiber) to evaluate any background ofendogenous FKBP heterodimerization, and Ad-199 (CEAVHH-FKBP, wt fiber)as a negative control (for complete virus list, see Table 1). 293 E4cells are infected with a multiplicity of infection (MOI) of 10, andmedia are replaced 4 hours after addition of virus. The cells aretreated with 500 nM rap or solvent control (EtOH) at 24 h p.i., and arecollected for lysis 36 h p.i. Total cell extract are used for IP.

To demonstrate that the FRB/FKBP interaction is biologically relevantand occurring on the surface of adenovirus particles, CoIP of theVHH-FKBP through non-fiber adenovirus capsid proteins from the lysatesof infected 293 E4 cells are performed. In addition, purification ofAd-177 and Ad-178 by CsCl gradient ultracentrifugation and anionexchange with and without rap is performed to see if the VHH-FKBP is(nonimmuno)precipitated/retained through these processes in the presenceof rap.

Rapamycin Induced Retargeting of Virus Tropism

The dimerization induced by rap on FKBP-retargeted viruses should enablethem to infect via disparate receptors based on the affinity of thetargeting moiety to a cellular receptor. That principle is demonstratedwith the Ad-177, Ad-178 viruses directed to CEA and EGFR respectively.Ad-177 and Ad-178 are used to infect 293 E4 cells and treated with 50015 nM rap or solvent control. Viruses are harvested from the media 48 hp.i. and directly used to infect a panel of cancer cell lines. Cellswere infected in duplicate and at different dilutions of the infectiousmedia (i.e.: undiluted, 2-fold, 4-fold, etc.). Infection is determinedby quantifying the number of GFP positive cells by FACS andhigh-throughput imaging.

The FKBP-ligand fusion can also be expressed ectopically in infectedcells to enable targeting of recombinant adenovirus with an insertedFRB-domain in the capsid. If an FKBP-ligand and dimerizing agent ispresent with the recombinant adenovirus, the targeted complex shouldassemble, even if the FKBP-ligand was not expressed from the adenovirusgenome. We will transiently express EGFRVHH-FKBP ectopically (from aplasmid), then infect cells with Ad-122. Ad-122 alone was not targetedin the presence of rapamycin, but in the additional presence ofEGFRVHH-FKBP, there was enhanced infection (FIG. 26). The ability toexpress the FKBP-ligand ectopically will enable rapid screening ofligand candidates or ligands in a library, without the need to assemblenew recombinant adenovirus genomes. For example, in a multi-well format,each well of cells could transiently express a pool of ligand-FKBPs,each would be infected with Ad-122, and the resulting viral supes(supernatant) could be applied to cells in culture to quantifyenhancement of targeting. The identity of the members in the ligand-FKBPpool can be further tested individually. Further, effective ligand-FKBPclones can be mutagenized and re-screened to enhance the effectivenessof targeted adenovirus infection.

Validate Retargeting Specificity

The effects observed from rapamycin-preparation of viruses should beconfirmed that the interaction is gained via interaction with theVHH-targeting moiety to verify the system. In the cases of viruses thatexhibit retargeting and different tropisms, specificity is verified byusing different VHH fusions, by knocking down CAR and the cellulartarget of the VHH via shRNA (e.g. EGFR knockdown for Ad-178), orblocking the cellular target with exogenous antibodies or VHH (e.g. addexcess exogenous EGFRVHH to block before Ad-178 infection). Alternativechemical-induced dimer systems such as orthogonal FRB/FKBP mutants thatutilize rap analogs (rapalogs) may also be necessary if endogenous mTORor FKBP interfere with virus component assembly. Alternatively otherdimerization systems could be explored (Hubbard, K. E., et al., Genes &Development, 24(16): p. 1695-1708 (2010)).

Rapalog Induced Retargeting of Virus Tropism

Since rapamycin may exhibit undesirable biological effects, such asgrowth and proliferation arrest (Jacinto E, Hall M N. Tor signalling inbugs, brain and brawn. Nat Rev Mol Cell Biol. 2003;4(2):117-26), anbiologically orthogonal molecule with the same heterodimerizingcapability is used to retarget adenovirus infection. The rapalog AP21967(FIG. 7) is able to form stable heterodimers with FKBP and a mutant FRBdomain (mTOR mutation T2098L), but not with the wt FRB domain (Bayle JH, Grimley J S, Stankunas K, Gestwicki J E, Wandless T J, Crabtree G R.Rapamycin analogs with differential binding specificity permitorthogonal control of protein activity. Chem Biol. 2006;13(1):99-107).The recombinant adenovirus encoding EGFRVHH-FKBP and the mutantFRB-modified fiber (Ad-220) was assembled and tested for targeting usingeither rapamycin or AP21967. Both rapamycin and AP21967 were able toretarget Ad-220, while the control virus was only targeted withrapamycin and not AP21967.

V. Material and Methods

Adsembly

Modified adenoviruses were made with the below referenced components.Gateway DONR vectors were employed. In the example of human Ad5, the E1module was obtained by PCR and inserted into the vector pDONR P1P4 usingSLIC. The pDONR P1P4 vector backbone including attL1 and attL4recombination sites was amplified using PCR and combined with the Ad5 E1module by SLIC. In order to generate an alternate counter-selectioncassette, vector pDONR P1P4 was modified. This vector backbone includingattP1 and attP4 recombination sites was amplified using PCR and combinedwith the PheS_(A294G) mutations and a Tetracycline resistance cassette(the pLac-Tet cassette from pENTR L3-pLac-Tet-L2) to create a new DONRvector. The attR1-PheS_(A294G)Tet(r)-attR4 fragment from the new DONRvector was then amplified by PCR and inserted into the Adsembly DESTvector. See “MultiSite Gateway® Pro Plus”, Cat #12537-100; and Sone, T.et al. J Biotechnol. 2008 Sep. 10; 136(3-4):113-21.

In the example of human Ad5, E3 module was inserted into the pDONR P5P3rvector by gateway BP reaction. The E4 module was inserted into pDONRP3P2 vector by gateway BP reaction. The attR5-ccdB-Cm(r)-attR2 fragmentfrom the pDONR P5P2 vector was amplified by PCR and inserted into theAdsembly DEST vector. See “MultiSite Gateway® Pro Plus”, Cat #12537-100;and Sone, T. et al. J Biotechnol. 2008 Sep. 10; 136(3-4):113-21.

The vector backbone for the Adsembly DEST vector is composed of partsfrom three different sources. The Amp(r) cassette and lacZ gene wasamplified from plasmid pUC19. This was combined with the p15A origin ofreplication, obtained from plasmid pSB3K5452002, part of theBioBricksiGEM 2007 parts distribution. The p15A ori, which maintainsplasmids at a lower (10-12) copy number is necessary to reduce E1toxicity. Lastly, in order to create a self-excising virus, themammalian expression cassette for the enzyme ISceI was PCR amplifiedfrom plasmid pAdZ5-CV5-E3+. This cassette was cloned into the vectorbackbone to create the vector called p15A-SceI. This is the vector usedto start genome assembly. In the example of human Ad5, the gene moduleswere all obtained from either DNA purified from wild type Ad5 virus orthe plasmid pAd/CMV/V5/DEST (Invitrogen).

Regarding the DEST vector in the example of human Ad5, the E2 and L3modules were inserted into plasmid p15A-SceI by 3-fragment SLIC. Thecounterselection marker expressing ccdB and Chlor(r) flanked by attR5and attR2 sites was obtained by PCR from plasmid pDONR P5P2. The secondcounterselection marker (PheS-Tet), was obtained by PCR from the vectorpDONR P1P4 PheS_(A294G)-Tet (see above). The two counter-selectionmarkers were inserted on the right (ccdB/Cm) and left (PheS/Tet) sidesof p15A-SceI E2-L4 by SLIC after cutting with unique restriction enzymesengineered to the ends of the E2 and L4 modules to create the DESTvector (pDEST E2-L5).

Regarding the multisite gateway entry vector containing adenoviral genemodules, in the example of human Ad5, the E1 module were inserted intopDONR P1P4 by SLIC. The E3 module was inserted into pDONR P5P3R bygateway BP reaction. The E4 module was inserted into pDONR P3P2 bygateway BP reaction.

Regarding Amp(r) cassette: plasmid pUC19, the p15A ori: plasmidpSB3K5-I52002 was part of the BioBricksiGEM 2007 parts distribution.Regarding the adenoviral gene modules, either the DNA purified from Ad5particles, or plasmid pAd/CMV/V5/DEST (Invitrogen). The DONR vectorspDONR P1P4, P5P2, P5P3R, P3P2 were received from Jon Chesnut(Invitrogen). The PheS gene was derived from DH5alpha bacterial genomicDNA and subsequently mutated by quick change to create the PheS_(A294G)mutant. Regarding the Tet(r) gene, the plasmid pENTR L3-pLac-Tet-L2 wasreceived from Jon Chesnut (Invitrogen).

Regarding an embodiment of the Adsembly method, 20 fmol of a dual DESTvector, typically containing a core module flanked by twocounterselection cassettes, is combined with 10 fmol of each remainingentry vector containing gene modules. In the example of Ad5, thisincludes combining 20 fmol of the E2-L3 dual DEST vector with 10 fmoleach of an E1 module entry vector, an E3 module entry vector, and an E4module entry vector. In some cases, increasing the amount of one or moreof the entry vectors may increase efficiency (e.g. using 50 fmol of theE1 module entry vector for Ad5). These vectors are combined with 2 μl ofLR Clonase II (Invitrogen) in a final volume of 10 μl. The reaction isincubated at 25° C. overnight (12-16 hours). The reaction is stopped bythe addition of 1 μl of proteinase K (Invitrogen) and incubation at 37°C. for 10 minutes. Five μl of the reaction is then transformed into highcompetency bacteria (>1e9 cfu/μg) that are sensitive to the ccdB geneproduct and plated onto YEG-C1 agar plates (as described in Kast, P.Gene, 138 (1994) 109-114; when using PheS_(A294G) counterselection) orother appropriate media for the counterselection used in the vector.Colonies are subsequently isolated and screened for complete genomes.Complete genomes are directly transfected into 293 E4 cells, resultingin infectious particles 5-9 days post-transfection.

Regarding PCRs, all PCRs were performed using the Phusion enzyme (NEB).PCRs to obtain the ADENOVIRAL GENE modules from Ad5 were performed with1×HF buffer, 200 μM each dNTP, 0.5 μM each primer, and 10 ng oftemplate. For the E2-L2 module, 3% DMSO was also added. Template waseither plasmid pAd/PL-DEST (Invitrogen; for E2-L2, L3-L4, and E4modules) or Ad5 genomic DNA (for E1 and E3 modules). PCR conditions wereas follows. E2-L2 and L3-L4: 98° C. 30 sec-10 cycles of 98° C. 10 sec,65° C. 30 sec (decrease temp 1° C. every 2 cycles), 72° C. 7 min-29cycles of 98° C. 10 sec, 60° C. 30 sec, 72° C. 8 min-72° C. 10 min-4° C.hold. E3: 98° C. 30 sec-10 cycles of 98° C. 10 sec, 70° C. 30 sec(decrease temp 0.5° C. every cycle), 72° C. 2 min 30 sec-25 cycles of98° C. 10 sec, 68° C. 30 sec, 72° C. 2 min 30 sec-72° C. 10 min-4° C.hold. E4: 98° C. 30 sec-6 cycles of 98° C. 10 sec, 63° C. 30 sec(decrease temp 0.5° C. every cycle), 72° C. 2 min-29 cycles of 98° C. 10sec, 60° C. 30 sec, 72° C. 2 min-72° C. 5 min-4° C. hold. Regardingobtaining viral genomic DNA from purified virus, up to 100 μl ofpurified virus is added to 300 μl of lysis buffer containing 10 mM TrispH 8, 5 mM EDTA, 200 mM NaCl, and 0.2% SDS. Mix is incubated at 60° C.for 5 min, followed by addition of 5 μl of proteinase K stock (˜20mg/mL) and further incubated at 60° C. for 1 hour. Samples are thenplaced on ice for 5 min, followed by spinning at 15K×g for 15 min.Supernatant is removed and added to an equal volume of isopropanol,mixed well, and spun at 15K×g for 15 min at 4° C. Pellet is washed with70% ethanol and re-spun for 15 min at 4° C. The pellet is dried andresuspended for use.

Regarding SLIC, linear fragments are exonuclease treated for 20 min atroom temp in the following 20 μl reaction: 50 mM Tris pH 8, 10 mM MgCl₂,50 m/mL BSA, 200 mM Urea, 5 mM DTT, and 0.5 μl T4 DNA polymerase. Thereaction is stopped by addition of 1 μl 0.5 M EDTA, followed byincubation at 75° C. for 20 min. An equal amount of T4-treated DNAs arethen mixed to around 20 μl in volume in a new tube. For SLIC combining 2fragments, 10 μl of each reaction is used. For SLIC combining 3fragments, 7 μl of each reaction is used.

Fragments are annealed by heating to 65° C. for 10 min, followed by aslow cool down decreasing the temperature 0.5° C. every 5 seconds downto 25° C. After annealing, 5 μl of the reaction is transformed andclones are screened.

Retargeted Virus Preparation

Regarding virus production, concentration and purification, 293 E4 cellsare infected with infectious particles, and approximately 48 hourspost-transfection when CPE is apparent, the cells are collected andisolated by centrifugation at 500×g for 5 minutes. The cells are lysedin TMN buffer (10 mM TrisCl pH 7.5, 1 mM MgCl₂, 150 mM NaCl) via 3×freeze/thaws, and the cell debris was removed by two rounds ofcentrifugation at 3K×g and 3.5K×g for 15 minutes. A cesium chloridegradient (0.5 g/mL) is used to band virus particles viaultracentrifugation at 37K×g for 18-24 hours. The band is collected anddialyzed in a 10 k MWCO Slide-A-Lyzer® dialysis cassette (ThermoScientific) in TMN with 10% glycerol overnight (12-18 h) at 4° C., thenstored at −80° C. The titer of the purified virus is determined versus atitered wildtype standard by a cell-based serial dilution infectionELISA with anti-adenovirus type 5 primary antibody (ab6982, Abcam), andImmunoPure anti-rabbit alkaline phosphatase secondary antibody (ThermoScientific).

Regarding insertion of the FRB domain of mTOR into the adenovirus fiber,the FRB domain was inserted into the H1-loop region of the fiber gene inthe Adsembly entry vector pENTR E3-L5 by SLIC. The 90aa FRB domain ofmTOR (amino acids Glu2025-Gln2114) was PCR amplified from pRK5 mTOR-myc(R. Shaw) for insertion into PCR amplified pENTR E3-L5 with endsflanking the adenovirus fiber H1-loop between Thr546 and Pro547 togenerate the resulting vector, pENTR E3-L5 (FRB-Fiber).

Regarding mutation of the FRB domain of mTOR to be specific for AP21967binding, the FRB domain was mutated using standard techniques in theAdsembly entry vector pENTR E3-L5 (FRB-Fiber) and pENTR E3-L5 (ΔRID,EGFRVHH-FKBP, FRB-Fiber). The residue Thr2098 (mTOR numbering) wasmutated to Leu. This resulted in the Adsembly entry vectors pENTR E3-L5(FRB*-Fiber) and pENTR E3-L5 (ΔRID, EGFRVHH-FKBP, FRB*-Fiber).

Regarding adenovirus-encoded fluorescent reporter for infection, thesequence for GFP was inserted 5′ of the adenovirus E1A gene to generatethe fusion described by Zhao, L. J. et al. J Biol Chem. 2006 Dec. 1;281(48):36613-23. The GFP gene was PCR amplified with the describedC-linker sequence for insertion into PCR amplified pENTR E1 with endsflanking the start codon of adenovirus E1A to generate the resultingplasmid, pENTR E1 (GFP-E1A).

Regarding expression of FKBP from virus genome, the FKBP sequence wasinserted by SLIC into pENTR E3-L5, replacing the adenovirus RIDα, RIDβ,and 14.7K genes. The FKBP gene was PCR amplified from pcDNA-FKBP12-Crluc(S. Gambhir) for insertion into PCR amplified pENTR E3-L5 and pENTRE3-L5 (FRB-Fiber) lacking the sequence from the start codon of RIDα tothe stop codon of 14.7 to generate the resulting vectors, pENTR E3-L5(ΔRID, FKBP) and pENTR E3-L5 (ΔRID, FKBP, FRB-Fiber). Alternative FKBPinsertion locations were constructed, but did not appear to lead toaccumulation of FKBP during infection via immunoblot (C-term IRES-drivenexpression on E1 transcript, C-term IRES-driven expression on fibertranscript, or Fiber-Furin2A-FKBP autocleavage sequence).

Regarding retargeting moiety genetic fusion with FKBP, 3D modeling inPyMol of FRB-Fiber in rapamycin-dependent complex with FKBP revealed anadvantage to fuse the targeting moiety to the N-terminus of FKBP. In thecase of the camelid antibody variable heavy chain (VHH) with EGFRbinding specificity (EGFRVHH), EGFRVHH was gene synthesized by BlueHeron Biotech, and inserted at the N-terminus of FKBP in pENTR E3-L5(ΔRID, FKBP) and pENTR E3-L5 (ΔRID, FKBP, FRB-Fiber) by SLIC with aGSGSGST linker sequence, to generate the plasmids pENTR E3-L5 (ΔRID,EGFRVHH-FKBP) and pENTR E3-L5 (ΔRID, EGFRVHH-FKBP, FRB-Fiber).

Retargeting Experiments

Regarding rapamycin-induced retargeting adenovirus infection via EGFR byEGFRVHH-FKBP, a virus was constructed using Adsembly with pENTR E1(GFP-E1A), pENTR E3-L5 (ΔRID, EGFRVHH-FKBP, FRB-Fiber), pENTR E4, andpDEST E2-L5, referred to hereafter as Ad-178, to infect a panel ofcancer cell lines. Ad-178 was used to infect 293 E4 cells at MOI 10.Twenty-four hours following the infection 50 nM rapamycin was added tothe medium. The concentration of rapamycin was optimized by testing arange of rapamycin concentrations with Ad-178 to infect MDA MB 453.Forty-eight hours following infection, the media containing infectiousparticles was collected and filtered through a 0.22 μm pore filter. Thefiltered media was used in serial dilution to infect a panel of cancercell lines in black-walled 96-well plates or 6-well plates, and virussimilarly prepared without the addition of rapamycin was used to infectan identical set of cells in parallel as the control. The media wasreplace 3 hours post-infection.

Regarding rapalog-induced retargeting adenovirus infection via EGFR byEGFRVHH-FKBP, a virus was constructed using Adsembly with pENTR E1(GFP-E1A), pENTR E3-L5 (ΔRID, EGFRVHH-FKBP, FRB*-Fiber), pENTR E4, andpDEST E2-L5, referred to hereafter as Ad5-220. Ad5-220 was used toinfect 293 E4 cells at MOI 10. Twenty-four hours following the infection100 nM AP21967 was added to the medium. Forty-eight hours followinginfection, the media containing infectious particles was collected andfiltered through a 0.22 μm pore filter. The filtered media was used toinfect cancer cell lines in 12-well plates, and viruses similarlyprepared with the addition of rapamycin or without the addition ofAP21967 were used to infect an identical set of cells in parallel ascontrols. The media was replaced 1 hour post-infection.

Regarding rapamycin-induced retargeting adenovirus infection withectopically expressed ligand-FKBP, 293 E4 cells were transientlytransfected with EGFRVHH-FKBP (or GFP as a control), and 24 hoursfollowing transfection, were infected with Ad-122 at MOI 10. Twenty-fourhours following the infection 100 nM rapamycin was added to the medium.Forty-eight hours following infection, the media containing infectiousparticles was collected and filtered through a 0.22 μm pore filter. Thefiltered media was used to infect MDA MB 231 cells seeded on 12-wellplates. The media was replaced 1 hour post-infection.

Regarding quantification of the infection efficiency, 96-well plateswere quantified by high-content imaging by counting % GFP-positive cellsusing IMAGEXPRESS™ software on an 25 IMAGEXPRESS™ Micro. The infectionefficiency of 6-well or 12-well plates was quantified by counting %GFP-positive cells by FACS using a FACScan (BD Biosciences).

Regarding the specificity of the EGFRVHH retargeted adenovirus to EGFR,the effective shRNA B sequence from Engelman, J. A. et al. J Clin Inv.2006 Oct. 2; 116(10):2695-706 was cloned under the control of an H1promoter in the pLentiX2 puro vector (Addgene), and used to generatelentivirus to mediate EGFR knockdown in MDA MB 453 breast cancer cells.MDA MB 453s were transduced with the anti-EGFR shRNA construct or acontrol lentivirus encoding an shRNA directed against the luciferasegene, and were selected under 2 μg/mL puromycin. Knockdown efficiencywas quantified by immunoblotting for EGFR in total protein. Theretargeting assay as described above was repeated on the selected MDA MB453 in 6-well plates, and the infection efficiencies were quantified byFACS using a FACScan (BD Biosciences).

VI. Tables

TABLE 1 Summary of designed novel adenoviruses. Ad-Serotype ComponentsAdenovirus E1 E2, E3 E4 L3 Ad-122 GFP- wt FRB-fiber wt (SEQ ID NO: 70)E1A Ad-177 GFP- wt ΔE3-RIDα, ΔE3-RIDβ, ΔE3- wt (SEQ ID NO: 71) E1A 14.7Kreplaced with CEAVHH- FKBP fusion; FRB-fiber Ad-178 GFP- wt ΔE3-RIDα,ΔE3-RIDβ, ΔE3- wt (SEQ ID NO: 72) E1A 14.7K replaced with EGFRVHH-FKBPfusion; FRB- fiber Ad-199 GFP- wt ΔE3-RIDα, ΔE3-RIDβ, ΔE3- wt (SEQ ID NO67) E1A 14.7K replaced with CEAVHH- FKBP fusion; wt fiber Ad-200 GFP- wtΔE3-RIDα, ΔE3-RIDβ, ΔE3- wt (SEQ ID NO: 68) E1A 14.7K replaced withEGFRVHH-FKBP fusion; wt fiber Ad-281 GFP- wt ΔE3-RIDα, ΔE3-RIDβ, ΔE3- wt(SEQ ID NO: 109) E1A 14.7K replaced with PA D4- FKBP fusion; FRB fiberAd-220 GFP- wt ΔE3-RIDα, ΔE3-RIDβ, ΔE3- wt (SEQ ID NO: 110) E1A 14.7Kreplaced with EGFRVHH-FKBP fusion; FRB (mTOR T2098L) fiber

TABLE 2 Examples of dimerizing agents (DA), dimerizing agent binders ofthe capsid dimerizing-agent binder conjugate (DABC) and dimerizing agentbinders of the ligand- dimerizing agent binder conjugate (DABL). DA DABCDABL rapamycin FRB FKBP12 AP21967 FRB (with FKBP12 mTOR T2098L mutation)abscisic acid (ABA) PYL1 AB1 2,4-dichlorophenoxyacetic acid (CFA) Tir1IAA7 inositol hexakisphosphate (IHP) indole-3-acetic acid (Auxin/IAA)

TABLE 3Examples of ligands included in the ligand-dimerizing agent binder conjugate and corresponding cell surfacereceptors bound by such ligands. Retargeting Uniprot AccessionUniprot Accession Element Number\Sequence Receptor Number Receptor Notesapelin Q9ULZ1 APLNR P35414 Widely expressed in brain, glial cells,(SEQ ID NO: 73) (SEQ ID NO: 10)astrocytes, neuronal subpopulations, spleen,thymus, ovary, small intestine, and colon. bradykinin P01042 BDKRB1,P30411 BDKRB1 is expressed in tissue injury, at sites (SEQ ID NO: 74) B2(SEQ ID NO: 11) of inflammation. B2 is ubiquitously expressed, P46663widespread in normal smooth muscle and (SEQ ID NO: 12) neurons.calcitonin P01258 CALCR P30988 Receptor found on osteoclasts.(SEQ ID NO: 75) (SEQ ID NO: 13) conantokin e.g. P07231 NMDAR1, Q05586Found upregulated in invasive tumor cells. peptides (SEQ ID NO: 76)2A, 2B, 2C, (SEQ ID NO: 14) 2D Q12879 (SEQ ID NO: 15) Q13224(SEQ ID NO: 16) Q14957 (SEQ ID NO: 17) O15399 (SEQ ID NO: 18)cholecystokinin P06307 CCKAR, P32238Receptor found in CNS and gastrointestinal (SEQ ID NO: 77) CCKBR(SEQ ID NO: 19) tract, upregulated in some colorectal and P32239pancreatic tumors. (SEQ ID NO: 20) EGF peptide, P01133 EGFR P00533Receptor ubiquitously expressed, upregulated in TGFα (SEQ ID NO: 78)(SEQ ID NO: 21) numerous tumors. P01135 (SEQ ID NO: 79) endothelinP05305 EDNRA P25101 Receptors present in blood vessels, nerves, and(SEQ ID NO: 80) (ETA), (SEQ ID NO: 22) brain tissue. EDNRB P24530 (ETB)(SEQ ID NO: 23) F3 peptide KDEPQRRSARLSAK Nucleolin P19338Receptor on cell surface of endothelial  PAPPKPEPKPKKAP (SEQ ID NO: 24)cells. AKK (SEQ ID NO: 81) Factor XI P03951 F11 receptor Q9Y624Receptor is an epithelial tight junction adhesion (SEQ ID NO: 82)(SEQ ID NO: 25) molecule. Fc fragment of FCAR P24071Receptor present on the surface of myeloid IgA (CD98) (SEQ ID NO: 26)lineage cells such as neutrophils, monocytes,macrophages, and eosinophils. Fc fragment of FCER1A, P12319Receptors bind alpha and gamma polypeptide IgE FCER1G (SEQ ID NO: 27)with respectively with high affinity. P20491 (SEQ ID NO: 28)Fc fragment of FCGR1A P12314 Receptor is monocyte/macrophage specific.IgG (CD64) (SEQ ID NO: 29) galanin P22466 GALR1, R2, P47211Receptors expressd in CNS and some solid (SEQ ID NO: 83) R3(SEQ ID NO: 30) tumors. O43603 (SEQ ID NO: 31) O60755 (SEQ ID NO: 32)gastric inhibitory P09681 GIPR P48546Receptors found on beta cells in pancreas. polypeptide (SEQ ID NO: 84)(SEQ ID NO: 33) (GIP) gastrin releasing P07492 GRPR P30550Highly expressed in pancreas, also in stomach, peptide (GRP),(SEQ ID NO: 85) (SEQ ID NO: 34) adrenal cortex, and brain. bombesinP21591 (SEQ ID NO: 86) glucagon (GCG) P01275 GCGR P47871Receptor found on hepatocytes. (SEQ ID NO: 87) (SEQ ID NO: 35)LyP-1 peptide CGNKRTRGC C1QBP Q07021Receptor normally found in mitochondria, but is (SEQ ID NO: 88)(SEQ ID NO: 36) on cell surface of lymphatic, myeloid, andcancer cells in tumors. neuromedin P08949 NMBR P28336Found expressed and functional in lung (SEQ ID NO: 89) (SEQ ID NO: 37)carcinoma cells, related to GRPR. parathyroid P01270 PTH1R, 2R Q03431Receptor in osteoblasts and kidney. hormone (SEQ ID NO: 90)(SEQ ID NO: 38) P49190 (SEQ ID NO: 39) poliovirus VP3, Q91UD0 PVR P15151Receptor establishes cell-cell junctions between TIGIT (SEQ ID NO: 91)(CD155) (SEQ ID NO: 40) epithelial cells. Q495A1 (SEQ ID NO: 92)prolactin P01236 PRLR P16471Receptors found in mammillary glands, ovaries, (SEQ ID NO: 93)(SEQ ID NO: 41) pituitary glands, heart, lung, thymus, spleen,liver, pancreas, kidney, adrenal gland, uterus,skeletal muscle, skin, and areas of CNS. protective antigen P13423ANTXR1 P58335 TEM8 found in umbilical vein endothelial cells (domain 4)(SEQ ID NO: 94) (TEM8), R2 (SEQ ID NO: 42)and tumor endothelial cells. CMG2 in prostate, (CMG2) Q9H6X2thymus, ovary, testis, pancreas, colon, heart, (SEQ ID NO: 43)kidney, lung, liver, peripheral blood leukocytes,placenta, skeletal muscle, small intestine, andspleen. Involved in angiogenesis. protein C P04070 EPCR Q9UNN8Receptor found on endothelial cells. (PROC) (SEQ ID NO: 95)(SEQ ID NO: 44) ricin B-chain P02879 terminalBeta-D-galactopyranoside moieties on cell (SEQ ID NO: 96) galactosesurface glycoproteins and glycolipids found on residues most cells.secretin P09683 SCTR P47872 Receptor ubiquitously expressed.(SEQ ID NO: 97) (SEQ ID NO: 45) shigatoxin B Q8HA13 CD77 Q9NPC4Receptor found in renal epithelial tissues, CNS subunit (SEQ ID NO: 98)(SEQ ID NO: 46) neurons and endothelium, pancreas cancer, colon cancer.tachykinin P20366 NK1R, P25103Receptor binds family of neuropeptides known peptides (SEQ ID NO: 99)K2R, K3R P21452 (SEQ ID NO: 47) as tachykinins. Q9UHF0 (SEQ ID NO: 48)(SEQ ID NO: 100) P29371 (SEQ ID NO: 49) tetanus toxin B- P04958 SV2A, 2BQ7L0J3 Receptors found on neuronal cells. (heavy) chain (SEQ ID NO: 101)(SEQ ID NO: 50) Q7L1I2 (SEQ ID NO: 51) thrombin (F2) P00734 F2R P25116Receptor has high affinity for activated (SEQ ID NO: 102)(SEQ ID NO: 52) thrombin, and is found mostly in smooth muscleand heart. thrombospondin- P07996 CD36, P16671CD36 found on platelets and 1 (TSP1) (SEQ ID NO: 103) CD47,(SEQ ID NO: 53) monocytes/macrophages. CD47 is broadly integrins Q08722distributed, abundant in some epithelia and the (SEQ ID NO: 54)brain, and has been found in ovarian tumors.TSP1 can bind to fibrinogen, fibronectin,laminin, type V collagen and integrins alpha-V/beta-1, alpha-V/beta-3 and alpha-IIb/beta-3. Transferrin, TfR P02787TFRC P02786 Receptor is found in endothelial cells and colon,binding peptides (SEQ ID NO: 104) (CD71), (SEQ ID NO: 55)and is constitutively endocytosed. It is TFR2 Q9UP52upregulated by cancer drug arabinoside (SEQ ID NO: 56) cytosine.vasoactive P01282 VIPR1, R2 P32241VPAC1 found in CNS, liver, lung, intestine, and intestinal peptide(SEQ ID NO: 105) (SEQ ID NO: 57) T-lymphocytes. VPAC2 found in CNS,P41587 pancreas, skeletal muscle, heart, kidney, adipose (SEQ ID NO: 58)tissue, testis, and stomach. VEGF P15692 VEGFR1, P17948VEGFR1 found in normal lung, placenta, liver, (SEQ ID NO: 106) R2, R3(SEQ ID NO: 59) kidney, heart, and brain. Specifically expressed P35968in vascular endothelial cells and peripheral (SEQ ID NO: 60)blood monocytes. VEGFR3 is expressed in P35916corneal epithelial cells and vascular smooth (SEQ ID NO: 61)muscle cells. von Willebrand P04275 (GPIbA, P07359Receptor complex found on platelets. factor (SEQ ID NO: 107) GPIbB,(SEQ ID NO: 62) GP9, and P13224 GP5) in (SEQ ID NO: 63) concert P14770(SEQ ID NO: 64) P40197 (SEQ ID NO: 65) scFvs Any*Can be designated by directed evolution of antibodies. VHH Any*Can be designated by directed evolution of antibodies.

VII. Embodiments

Embodiment 1. A recombinant nucleic acid encoding a capsid-dimerizingagent binder conjugate and a ligand-dimerizing agent binder conjugate.

Embodiment 2. The recombinant nucleic acid of embodiment 1, wherein saidcapsid-dimerizing agent binder conjugate comprises a capsid protein anda dimerizing agent binder.

Embodiment 3. The recombinant nucleic acid of embodiment 2, wherein saidcapsid protein is operably linked to said dimerizing agent binder.

Embodiment 4. The recombinant nucleic acid of embodiment 3, wherein saidcapsid protein is an adenoviral capsid protein.

Embodiment 5. The recombinant nucleic acid of embodiment 4, wherein saidadenoviral capsid protein is a fiber protein.

Embodiment 6. The recombinant nucleic acid of embodiment 3, wherein saiddimerizing agent binder is a FRB protein.

Embodiment 7. The recombinant nucleic acid of embodiment 1, wherein saidligand-dimerizing agent binder conjugate comprises a ligand and adimerizing agent binder.

Embodiment 8. The recombinant nucleic acid of embodiment 7, wherein saidligand is operably linked to said dimerizing agent binder.

Embodiment 9. The recombinant nucleic acid of embodiment 7, wherein saidligand is capable of binding a cell.

Embodiment 10. The recombinant nucleic acid of embodiment 9, whereinsaid cell is a tumor cell.

Embodiment 11. The recombinant nucleic acid of embodiment 7, whereinsaid ligand is an antibody.

Embodiment 12. The recombinant nucleic acid of embodiment 11, whereinsaid antibody is a single domain antibody.

Embodiment 13. The recombinant nucleic acid of embodiment 7, whereinsaid dimerizing agent binder is an immunophilin protein.

Embodiment 14. The recombinant nucleic acid of embodiment 13, whereinsaid immunophilin protein is a FKBP protein.

Embodiment 15. The recombinant nucleic acid of embodiment 14, whereinsaid FKBP protein is a human FKBP protein.

Embodiment 16. The recombinant nucleic acid of embodiment 15, whereinsaid human FKBP protein is FKBP12.

Embodiment 17. A recombinant adenovirus comprising a recombinant nucleicacid of one of embodiments 1-16.

Embodiment 18. The recombinant adenovirus of embodiment 17, wherein saidadenovirus is a replication incompetent adenovirus.

Embodiment 19. The recombinant adenovirus of embodiment 17, wherein saidadenovirus is a replication competent adenovirus.

Embodiment 20. A recombinant adenovirus comprising a capsid-dimerizingagent binder conjugate.

Embodiment 21. The recombinant adenovirus of embodiment 20, wherein saidcapsid-dimerizing agent binder conjugate is bound to a dimerizing agent.

Embodiment 22. The recombinant adenovirus of embodiment 21, wherein saiddimerizing agent is a compound.

Embodiment 23. The recombinant adenovirus of embodiment 22, wherein saidcompound is rapamycin.

Embodiment 24. The recombinant adenovirus of embodiment 21, wherein saiddimerizing agent is an anti-cancer drug.

Embodiment 25. The recombinant adenovirus of embodiment 21, wherein saiddimerizing agent is further bound to a ligand-dimerizing agent binderconjugate.

Embodiment 26. A cell comprising a recombinant adenovirus of any one ofembodiments 20-25.

Embodiment 27. A method of forming an adenoviral cancer cell targetingconstruct, said method comprising: (i) infecting a cell with arecombinant adenovirus of embodiment 17, thereby forming an adenoviralinfected cell; (ii) allowing said adenoviral infected cell to expresssaid recombinant nucleic acid, thereby forming a ligand-dimerizing agentbinder conjugate and a recombinant adenovirus comprising acapsid-dimerizing agent binder conjugate; (iii) contacting saidrecombinant adenovirus and said ligand-dimerizing agent binder conjugatewith a dimerizing agent; (iv) allowing said recombinant adenovirus andsaid ligand-dimerizing agent binder conjugate to bind to said dimerizingagent, thereby forming said adenoviral cancer cell targeting construct.

Embodiment 28. A method of targeting a cell, said method comprisingcontacting a cell with a recombinant adenovirus of any one ofembodiments 20-25.

Embodiment 29. The method of embodiment 28, wherein said cell is acancer cell.

Embodiment 30. A method of targeting a cancer cell in a cancer patient,said method comprising: (i) administering to a cancer patient arecombinant adenovirus of embodiment 17; (ii) allowing said recombinantadenovirus to infect a cell in said cancer patient, thereby forming anadenoviral infected cell; (iii) allowing said adenoviral infected cellto express said recombinant nucleic acid, thereby forming aligand-dimerizing agent binder conjugate and a recombinant adenoviruscomprising a capsid-dimerizing agent binder conjugate; (iv)administering to said cancer patient a dimerizing agent; (v) allowingsaid recombinant adenovirus and said ligand-dimerizing agent binderconjugate to bind to said dimerizing agent, thereby forming anadenoviral cancer cell targeting construct; (vi) allowing saidadenoviral cancer cell targeting construct to bind to a cancer cell,thereby targeting said cancer cell in said cancer patient.

Embodiment 31. The method of embodiment 30, wherein said cell is acancer cell.

Embodiment 32. The method of embodiment 30, wherein said cell is anon-cancer cell.

Embodiment 33. A method of targeting a cell, said method comprising: (i)contacting a first cell with a recombinant adenovirus of embodiment 17;(ii) allowing said recombinant adenovirus to infect said first cell,thereby forming an adenoviral infected cell; (iii) allowing saidadenoviral infected cell to express said recombinant nucleic acid,thereby forming a ligand-dimerizing agent binder conjugate and arecombinant adenovirus comprising a capsid-dimerizing agent binderconjugate; (iv) contacting said ligand-dimerizing agent binder conjugateand said recombinant adenovirus with a dimerizing agent; (v) allowingsaid recombinant adenovirus and said ligand-dimerizing agent binderconjugate to bind to said dimerizing agent, thereby forming anadenoviral cell targeting construct; (vi) allowing said adenoviral celltargeting construct to bind to a second cell, thereby targeting saidcell.

Embodiment 34. The method of embodiment 33, wherein said first cell andsaid second cell form part of an organism.

Embodiment 35. The method of embodiment 33, wherein said first cell andsaid second cell form part of tissue culture vessel.

1. A recombinant nucleic acid encoding a capsid-dimerizing agent binderconjugate and a ligand-dimerizing agent binder conjugate, wherein thecapsid-dimerizing agent binder conjugate comprises an adenoviral fiberprotein and a FRB protein inserted into the H1 loop of the adenoviralfiber protein, and the ligand-dimerizing agent binder conjugatecomprises a ligand and a FKBP protein.
 2. The recombinant nucleic acidof claim 1, wherein the ligand is capable of binding a tumor cell. 3.The recombinant nucleic acid of claim 2, wherein the ligand is anantibody.
 4. The recombinant nucleic acid of claim 3, wherein theantibody is a single domain antibody.
 5. The recombinant nucleic acid ofclaim 1, wherein the FRB protein is 90 amino acids in length.
 6. Therecombinant nucleic acid of claim 1, wherein the FRB protein comprises awild-type mTOR FRB domain.
 7. The recombinant nucleic acid of claim 1,wherein the FRB protein is encoded by the nucleotide sequence of SEQ IDNO:
 69. 8. The recombinant nucleic acid of claim 1, wherein the FRBprotein comprises a mutant mTOR FRB domain.
 9. The recombinant nucleicacid of claim 8, comprising SEQ ID NO:
 110. 10. The recombinant nucleicacid of claim 1, wherein the FKBP protein is a human FKBP protein. 11.The recombinant nucleic acid of claim 10, wherein the human FKBP proteinis FKBP12.
 12. A recombinant adenovirus comprising the recombinantnucleic acid of claim
 1. 13. A recombinant adenovirus comprising acapsid-dimerizing agent binder conjugate, wherein the capsid-dimerizingagent binder conjugate comprises an adenoviral fiber protein and a FRBprotein inserted into the H1 loop of the adenoviral fiber protein. 14.The recombinant adenovirus of claim 13, wherein the capsid-dimerizingagent binder conjugate is bound to a dimerizing agent.
 15. Therecombinant adenovirus of claim 14, wherein the dimerizing agent isfurther bound to a ligand-dimerizing agent binder conjugate.
 16. Therecombinant adenovirus of claim 14, wherein the dimerizing agent israpamycin or a rapalog.
 17. The recombinant adenovirus of claim 13,wherein the FRB protein is 90 amino acids in length.
 18. The recombinantadenovirus of claim 13, wherein the FRB protein comprises a wild-typemTOR FRB domain.
 19. The recombinant adenovirus of claim 18, wherein theFRB protein is encoded by the nucleotide sequence of SEQ ID NO:
 69. 20.The recombinant adenovirus of claim 13, wherein the FRB proteincomprises a mutant mTOR FRB domain.